Atropisomers of asymmetric xanthene fluorescent dyes and methods of DNA sequencing and fragment analysis

ABSTRACT

Atropisomeric energy-transfer dye compounds are disclosed. A variety of molecular biology applications utilize atropisomeric xanthene fluorescent dyes as labels for substrates such as nucleotides, nucleosides, polynucleotides, polypeptides and carbohydrates. Methods include DNA sequencing, DNA fragment analysis, PCR, SNP analysis, oligonucleotide ligation, amplification, minisequencing, and primer extension.

This aplication is a divisional of application Ser. No. 10/227,058,filed on Aug. 21, 2002 now U.S. Pat. No. 6,649,769 which is a divisionalof application Ser. No. 09/704,966 filed on Nov. 1, 2000, now U.S. Pat.No. 6,448,407, all of which are incorporated herein by reference.

I. FIELD OF THE INVENTION

The invention relates to certain atropisomer forms of asymmetricxanthene fluorescent dyes and the field of nucleic acid sequencing andanalysis with fluorescent dye-labelled reagents.

II. BACKGROUND OF THE INVENTION

Methods of analyzing fluorescent-labelled biomolecules after separatingbased on size- or charge are central to molecular biology. Examples ofmethods utilizing fluorescent-labelled nucleic acids include automatedDNA sequencing, oligonucleotide probe methods, detection ofpolymerase-chain-reaction products, immunoassays, and the like. In thecase of multi-color automated DNA sequencing, labelled nucleic acidfragments of varying size are separated by electrophoresis, typically ina single electrophoresis lane, channel, or capillary. Employing thesemethods, automated four-color Sanger-type DNA sequencing has enabledentire genome characterization at the molecular level.

Stereochemical purity is of importance in the field of pharmaceuticals,where 12 of the 20 most prescribed drugs exhibit chirality (U.S. Pat.No. 6,075,024). A case in point is provided by the L-form of thebeta-adrenergic blocking agent, R(−) albuterol, which is known to be 100times more potent than the D-enantiomer (U.S. Pat. No. 5,760,090).Furthermore, optical purity is important since certain isomers mayactually be deleterious rather than simply inert.

Atropisomers are stereoisomeric conformations of a molecule whoseinterconversion is slow enough to allow separation and isolation underpredetermined conditions (McGraw-Hill Dictionary of Chemical Terms,(1984), S. Parker, Ed., p. 36). The energy barrier to thermalracemization may be determined by the steric hindrance to free rotationof one or more bonds forming a chiral axis. Certain biaryl compoundsexhibit atropisomerism where rotation around an intraannular bondlacking C₂ symmetry is restricted. The free energy barrier forenantiomerization is a measure of the stability of the intraannular bondwith respect to rotation. Optical and thermal excitation can promoteracemization, dependent on electronic and steric factors (Tetreau (1982)Nouv. Jour. de Chimie, 6:461–65).

Ortho-substituted biphenyl compounds may exhibit this type ofconformational, rotational isomerism known as atropisomerism (Eliel, E.and Wilen, S. (1994) Stereochemistry of Organic Compounds, John Wiley &Sons, Inc., pp. 1142–55). Such biphenyls are enantiomeric, chiralatropisomers where the sp2-sp2 carbon-carbon, intraannular bond betweenthe phenyl rings has a sufficiently high energy barrier to freerotation, and where substituents X≠Y and U≠V render the moleculeasymmetric. The steric interaction of X—U, X—V, and/or Y—V, Y—U is largeenough to make the planar conformation an energy maximum. Two nonplanar,axially chiral enantiomers, shown below, then exist as atropisomers whentheir interconversion is slow enough such that they can be isolated freeof each other. By one definition, atropisomerism is defined to existwhere the isomers have a half-life t½ of at least 1000 seconds, which isa free energy barrier of 22.3 kcal mol⁻¹ (93.3 kJ mol⁻¹) at 300K (Oki,M. (1983) “Recent Advances in Atropisomerism,” Topics inStereochemistry, 14:1). Bold lines and dashed lines in the figures shownbelow indicate those moieties, or portions of the molecule, which aresterically restricted due to a rotational energy barrier. Boldedmoieties exist orthoganally above the plane and dashed moieties existorthogonally below the plane of the rest of the molecule.

Xanthene dyes have important applications as detectable fluorescentlabels of nucleic acids (U.S. Pat. Nos. 5,188,934; 5,654,442; 5,885,778;6,096,723; 6,020,481; 5,863,727; 5,800,996; 5,945,526; 5,847,162;6,025,505; 6,008,379; 5,936,087; 6,015,719). Xanthene compoundscontaining an asymmetric biannular bond can exist in stableatropisomeric forms. Conjugates of atropisomeric xanthene compounds andchiral substrates, such as nucleotides, polynucleotides, polypeptides,and carbohydrates, form diastereomers. These diastereomeric conjugatescan separate under certain conditions, such as electrophoresis,chromatography, and other methods. Separation of diastereomers canhinder detection by display of double peaks or bands, i.e. “peakdoubling”. Thus, atropisomerically enriched or purified forms ofxanthene dyes are important as labels for methods based on separationand detection of analytes.

III. SUMMARY

The present invention is directed towards atropisomerically-enriched andsubstantially pure atropisomers of asymmetric xanthene compounds asnovel compositions. The invention also includes methods for isolation,labelling, and detecting labelled compositions.

In a first aspect, the invention includes substantially pure atropisomercompounds having the structure II:

wherein positions R¹, R⁴, R⁵, R¹¹, R¹³, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, Z¹, orZ² may be substituted with substituents. At least one substituent may bea linking moiety. One or more rings may be fused on the ring structureII.

Another aspect of the invention includes energy-transfer dye compoundscomprising a donor dye capable of absorbing light at a first wavelengthand emitting excitation energy in response thereto; an acceptor dyecapable of absorbing the excitation energy emitted by the donor dye andfluorescing at a second wavelength in response; and a linker for linkingthe donor dye and the acceptor dye; wherein at least one of the donordye and acceptor dye is a substantially pure atropisomer of a xanthenecompound.

Another aspect of the invention is labelled substrates, includingnucleoside, nucleotides, polynucleotides, and polypeptides wherein thelabel is a substantially pure atropisomer of a xanthene compound or anenergy-transfer dye comprising a substantially pure atropisomer of axanthene compound.

Another aspect of the invention is labelling reagents, includingphosphoramidite and active ester linking moieties of a substantiallypure atropisomer of a xanthene compound, which form covalent attachmentswith substrates and methods of labelling substrates with the reagents.

Another aspect of the invention is methods for forming a labelledsubstrate comprising the step of reacting a substrate with the linkingmoiety of a substantially pure atropisomer of a xanthene compound or anenergy-transfer dye comprising a substantially pure atropisomer of axanthene compound.

Another aspect of the invention is methods for separating atropisomersof xanthene compounds by forming diastereomers with substantiallyenantiomerically pure compounds, and separating the diastereomers. Thediastereomers may be converted to substantially pure atropisomers ofxanthene compounds.

Another aspect of the invention is methods for separating a mixture oflabelled substrates wherein the labels are comprised of a substantiallypure atropisomer of a xanthene compound or an energy-transfer dyecomprising a substantially pure atropisomer of a xanthene compound. Thelabelled substrates may be primer extension polynucleotide fragments.The labelled substrates may be separated by electrophoresis,chromatography, or other separation technique. The mixture of labelledpolynucleotides may be formed from a labelled primer or a labelledterminator. The labelled substrates may be detected by fluorescencedetection.

Another aspect of the invention is methods of generating a labelledprimer extension product by extending a primer-target hybrid with anenzymatically-incorporatable nucleotide. The primer or the nucleotidemay be labelled with a substantially pure atropisomer of a xanthenecompound or an energy-transfer dye comprising a substantially pureatropisomer of a xanthene compound.

Another aspect of the invention is methods of polynucleotide sequencingby forming a mixture of four classes of polynucleotides where each classis labelled at the 3′ terminal nucleotide with a substantially pureatropisomer of a xanthene compound or an energy-transfer dye comprisinga substantially pure atropisomer of a xanthene compound, and the labelsare spectrally resolvable. The polynucleotides are separated by size.

Another aspect of the invention is methods of oligonucleotide ligationby annealing two probes to a target sequence and forming aphosphodiester bond between the 5′ terminus of one probe and the 3′terminus of the other probe wherein one or both probes are labelled witha substantially pure atropisomer of a xanthene compound or anenergy-transfer dye comprising a substantially pure atropisomer of axanthene compound.

Another aspect of the invention is methods of amplification by annealingtwo or more primers to a target polynucleotide and extending the primersby a polymerase and a mixture of enzymatically-extendable nucleotideswherein at least one of the primers or one of the nucleotides islabelled with a substantially pure atropisomer of a xanthene compound oran energy-transfer dye comprising a substantially pure atropisomer of axanthene compound.

Another aspect of the invention is kits of reagents including asubstantially pure atropisomer of a xanthene compound or anenergy-transfer dye comprising a substantially pure atropisomer of axanthene compound.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a shows the reaction of C-1 aminomethyl, C-19 carboxy fluoresceinand (−) Menthyl chloroformate to give menthyl carbamate diastereomers 1aand 1b.

FIG. 1 b shows reverse phase HPLC analysis of preparative HPLC fractionsin the separation of menthyl carbamate diastereomers 1a and 1b.Fractions #1 and #2 show pure 1a. Fraction #3 shows a mixture of 1a and1b. Fractions #4 and #5 show pure 1b.

FIG. 2 a shows the reactions of: (i) menthyl carbamate diastereomer 1awith sulfuric acid to hydrolyze the menthyl carbamate group to giveatropisomer amine 2a, (ii) amidation of 2a with ethyl trifluoroacetateto give atropisomer trifluoroacetamide 3a, (iii) menthyl carbamatediastereomer 1b with sulfuric acid to hydrolyze the menthyl carbamategroup to give atropisomer amine 2b, and (iv) amidation of 2b with ethyltrifluoroacetate to give atropisomer trifluoroacetamide 3b.

FIG. 2 b shows an HPLC chromatogram of the racemic mixture ofatropisomers 2a and 2b under chiral HPLC conditions.

FIG. 2 c shows an HPLC chromatogram of the purified atropisomer 2a underchiral HPLC conditions.

FIG. 3 shows the reactions of: (i) atropisomer trifluoroacetamide 3awith N-hydroxysuccinimide and DAE carbodiimide HCl salt to giveatropisomeric NHS ester 4a, and (ii) atropisomer trifluoroacetamide 3bwith N-hydroxysuccinimide and DAE carbodiimide HCl salt to giveatropisomeric NHS ester 4b.

FIG. 4 shows the synthesis of2-[(2-Fmoc-aminoethoxy)(hydroxyphosphoryl)oxy]acetic acid NHS 5.

FIG. 5 shows the synthesis of propargylphosphorylamino-ddATP 11.

FIG. 6 shows the synthesis ofaminomethyl-FAM-propargylphosphorylamino-ddATP 15.

FIG. 7 shows the synthesis of tricyclic amine 20.

FIG. 8 shows the synthesis of NHS-rhodamine dye 17.

FIG. 9 shows the structure of energy-transfer ddATP terminator 25.

FIG. 10 shows the synthesis of bis-trifluoroacetamide rhodamine NHS 27.

FIG. 11 shows the synthesis ofaminomethylbenzamide-aminomethyl-FAM-propargylethoxyamino-ddTTP 32.

FIG. 12 shows the structure of energy-transfer ddTTP terminator 26.

FIG. 13 a shows DNA sequencing of pGEM with phosphate-linker,energy-transfer terminator ddATP 25, fragment size 119–242 bp.

FIG. 13 b shows DNA sequencing of pGEM with phosphate-linker,energy-transfer terminator ddATP 25, fragment size 148–205 bp.

FIG. 13 c shows DNA sequencing of pGEM with sulfonate-linker,energy-transfer terminator ddATP 33, fragment size 148–242 bp.

FIG. 13 d shows DNA sequencing of pGEM with energy-transfer terminatorddGTP 34, fragment size 24–99 bp.

FIG. 14 shows the structure of sulfonate-linker, energy-transferterminator ddATP 33.

FIG. 15 shows the structure of energy-transfer terminator ddGTP 34.

V. DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings. While the invention will be described in conjunction with theillustrated embodiments, it will be understood that they are notintended to limit the invention to those embodiments. On the contrary,the invention is intended to cover all alternatives, modifications, andequivalents, which may be included within the scope of the claimedinvention.

V.1 Definitions

Stereochemical definitions and conventions used herein generally followMcGraw-Hill Dictionary of Chemical Terms (1984) S. P. Parker, Ed.,McGraw-Hill Book Company, New York and Stereochemistry of OrganicCompounds (1994); and Eliel, E. and Wilen, S., John Wiley & Sons, Inc.,New York. Many organic compounds exist in optically active forms, i.e.,they have the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L or R and Sare used to denote the absolute configuration of the molecule about itschiral center(s). The prefixes d and 1 or (+) and (−) are employed todesignate the sign of rotation of plane-polarized light by the compound,with (−) or 1 meaning that the compound is levorotatory. A compoundprefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these compounds, called stereoisomers, are identical exceptthat they are mirror images of one another. A specific stereoisomer mayalso be referred to as an enantiomer, and a mixture of such isomers isoften called an enantiomeric mixture. A 50:50 mixture of enantiomers isreferred to as a racemic mixture or a racemate. The terms “racemicmixture” and “racemate” refer to an equimolar mixture of twoenantiomeric species, devoid of optical activity.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

The term “atropisomer” refers to a stereoisomer resulting fromrestricted rotation about single bonds where the rotation barrier ishigh enough to permit isolation of the isomeric species. Atropisomersare enantiomers without a single asymmetric atom. Atropisomers areconformational stereoisomers which occur when rotation about a singlebond in the molecule is prevented, or greatly slowed, as a result ofsteric interactions with other parts of the molecule and thesubstituents at both ends of the single bond are unsymmetrical.

The terms “substantially pure atropisomer” and “substantially free ofits stereoisomer” mean that the composition contains at least 90% byweight of one atropisomer, and 10% by weight or less of thestereoisomeric atropisomer.

The term “atropisomerically enriched” means that the composition is agreater proportion or percentage of one of the atropisomers of thexanthene compound, in relation to the other atropisomer.

“Nucleobase” means a nitrogen-containing heterocyclic moiety capable offorming Watson-Crick hydrogen bonds in pairing with a complementarynucleobase or nucleobase analog, e.g. a purine, a 7-deazapurine, or apyrimidine. Typical nucleobases are the naturally occurring nucleobasesadenine, guanine, cytosine, uracil, thymine, and analogs of thenaturally occurring nucleobases, e.g. 7-deazaadenine, 7-deazaguanine,7-deaza-8-azaguanine, 7-deaza-8-azaadenine (Kutyavin, U.S. Pat. No.5,912,340), inosine, nebularine, nitropyrrole, nitroindole,2-aminopurine, 2,6-diaminopurine, hypoxanthine, pseudouridine,pseudocytosine, pseudoisocytosine, 5-propynylcytosine, isocytosine,isoguanine, 7-deazaguanine, 2-thiopyrimidine, 6-thioguanine,4-thiothymine, 4-thiouracil, O⁶-methylguanine, N⁶-methyladenine,O⁴-methylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil,4-methyl-indole, and ethenoadenine (Fasman (1989) Practical Handbook ofBiochemistry and Molecular Biology, pp. 385–394, CRC Press, Boca Raton,Fla.).

“Nucleoside” means a compound consisting of a nucleobase linked to theC-1′ carbon of a ribose sugar. The ribose may be substituted orunsubstituted. Substituted ribose sugars include, but are not limitedto, those riboses in which one or more of the carbon atoms, e.g., the3′-carbon atom, is substituted with one or more of the same or different—R, —OR, —NRR or halogen groups, where each R is independently hydrogen,C₁–C₆ alkyl or C₅–C₁₄ aryl. Riboses include ribose, 2′-deoxyribose,2′,3′-dideoxyribose, 3′-haloribose, 3′-fluororibose, 3′-chlororibose,3′-alkylribose, e.g. 2′-O-methyl, 4′-α-anomeric nucleotides,1′-α-anomeric nucleotides, and 2′-4′-linked and other “locked”, bicyclicsugar modifications (Imanishi WO 98/22489; Imanishi WO 98/39352; WengelWO 99/14226). When the nucleobase is purine, e.g. A or G, the ribosesugar is attached to the N⁹-position of the nucleobase. When thenucleobase is pyrimidine, e.g. C, T or U, the pentose sugar is attachedto the N¹-position of the nucleobase (Kornberg and Baker, (1992) DNAReplication, 2^(nd) Ed., Freeman, San Francisco, Calif.).

“Nucleotide” means a phosphate ester of a nucleoside, as a monomer unitor within a nucleic acid. Nucleotides are sometimes denoted as “NTP”, or“dNTP” and “ddNTP” to particularly point out the structural features ofthe ribose sugar. “Nucleotide 5′-triphosphate” refers to a nucleotidewith a triphosphate ester group at the 5′ position. The triphosphateester group may include sulfur substitutions for the various oxygens,e.g. α-thio-nucleotide 5′-triphosphates.

As used herein, the terms “oligonucleotide” and “polynucleotide” areused interchangeably and mean single-stranded and double-strandedpolymers of nucleotide monomers, including 2′-deoxyribonucleotides (DNA)and ribonucleotides (RNA) linked by internucleotide phosphodiester bondlinkages, or internucleotide analogs, and associated counter ions, e.g.,H⁺, NH₄ ⁺, trialkylammonium, Mg²⁺, Na⁺ and the like. A polynucleotidemay be composed entirely of deoxyribonucleotides, entirely ofribonucleotides, or chimeric mixtures thereof. Polynucleotides may becomprised of internucleotide, nucleobase and sugar analogs.Polynucleotides typically range in size from a few monomeric units, e.g.5–40, when they are frequently referred to as oligonucleotides, toseveral thousands of monomeric nucleotide units. Unless denotedotherwise, whenever a polynucleotide sequence is represented, it will beunderstood that the nucleotides are in 5′ to 3′ order from left to rightand that “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G”denotes deoxyguanosine, and “T” denotes thymidine, unless otherwisenoted.

“Attachment site” means a site on a label or a substrate, such as anoligonucleotide, which is covalently attached to a linker.

“Linker” means a chemical moiety comprising a covalent bond or a chainof atoms that covalently attaches a label to a polynucleotide, or onelabel to another.

“Linking moiety” means a chemically reactive group, substituent ormoiety, e.g. a nucleophile or electrophile, capable of reacting withanother molecule to form a covalent bond, or linkage.

“Alkyl” means a saturated or unsaturated, branched, straight-chain,branched, or cyclic hydrocarbon radical derived by the removal of onehydrogen atom from a single carbon atom of a parent alkane, alkene, oralkyne. Typical alkyl groups consist of 1–12 saturated and/orunsaturated carbons, including, but not limited to, methyl, ethyl,propyl, butyl, and the like.

“Alkoxy” means —OR where R is (C₁–C₆) alkyl.

“Alkyldiyl” means a saturated or unsaturated, branched, straight chainor cyclic hydrocarbon radical of 1–20 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkane, alkeneor alkyne. Typical alkyldiyl radicals include, but are not limited to,1,2-ethyldiyl, 1,3-propyldiyl, 1,4-butyldiyl, and the like.

“Aryl” means a monovalent aromatic hydrocarbon radical of 6–20 carbonatoms derived by the removal of one hydrogen atom from a single carbonatom of a parent aromatic ring system. Typical aryl groups include, butare not limited to, radicals derived from benzene, substituted benzene,naphthalene, anthracene, biphenyl, and the like.

“Aryldiyl” means an unsaturated cyclic or polycyclic hydrocarbon radicalof 6–20 carbon atoms having a conjugated resonance electron system andat least two monovalent radical centers derived by the removal of twohydrogen atoms from two different carbon atoms of a parent arylcompound.

“Substituted alkyl”, “substituted alkyldiyl”, “substituted aryl” and“substituted aryldiyl” mean alkyl, alkyldiyl, aryl and aryldiylrespectively, in which one or more hydrogen atoms are each independentlyreplaced with another substituent. Typical substituents include, but arenot limited to, —X, —R, —O⁻, —OR, —SR, —S⁻, —NR₂, —NR₃, ═NR, —CX₃, —CN,—OCN, —SCN, —NCO, —NCS, —NO, —NO₂, ═N₂, —N₃, NC(O)R, —C(O)R,—C(O)NRR—S(O)₂O⁻, —S(O)₂OH, —S(O)₂R, —OS(O)₂OR, —S(O)₂NR, —S(O)R,—OP(O)O_(P(O)O) ₂RR—P(O)(O⁻)₂, —P(O)(OH)₂, —C(O)R, —C(O)X, —C(S)R,—C(O)OR, —C(O)O⁻, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR,—C(NR)NRR, where each X is independently a halogen and each R isindependently —H, alkyl, aryl, heterocycle, or linking group.

“Internucleotide analog” means a phosphate ester analog of anoligonucleotide such as: (i) alkylphosphonate, e.g. C₁–C₄alkylphosphonate, especially methylphosphonate; (ii) phosphoramidate;(iii) alkylphosphotriester, e.g. C₁–C₄ alkylphosphotriester; (iv)phosphorothioate; and (v) phosphorodithioate. Internucleotide analogsalso include non-phosphate analogs wherein the sugar/phosphate subunitis replaced by an a non-phosphate containing backbone structure. Onetype of non-phosphate oligonucleotide analogs has an amide linkage, suchas a 2-aminoethylglycine unit, commonly referred to as PNA (Nielsen(1991) “Sequence-selective recognition of DNA by strand displacementwith a thymidine-substituted polyamide”, Science 254:1497–1500).

The terms “target sequence” and “target polynucleotide” mean apolynucleotide sequence that is the subject of hybridization with acomplementary polynucleotide, e.g., a primer or probe. The sequence canbe composed of DNA, RNA, an analog thereof, including combinationsthereof.

The term “label”, as used herein, means any moiety which can be attachedto a substrate, e.g., an oligonucleotide, nucleotide or nucleotide5′-triphosphate, and that functions to: (i) provide a detectable signal;(ii) interact with a second label to modify the detectable signalprovided by the first or second label, e.g. FRET; (iii) stabilizehybridization, i.e. duplex formation; (iv) affect mobility, e.g.electrophoretic mobility or cell-permeability, by charge,hydrophobicity, shape, or other physical parameters, or (v) provide acapture moiety, e.g., affinity, antibody/antigen, or ionic complexation.

“Heterocycle” means a molecule with a ring system in which one or morering atoms have been replaced with a heteroatom, e.g. nitrogen, oxygen,and sulfur.

“Electron-deficient nitrogen heterocycle” is a monovalentelectron-deficient nitrogen heterocycle derived by the removal of onehydrogen atom from a single atom of the ring system to join theheterocycle as a substituent to the fluorescein dyes of the invention(Joule, Heterocyclic Chemistry, 3rd Ed., Stanley Thornes Publisher,Ltd., Cheltenham, U.K. (1998); Acheson, R., An Introduction to theChemistry of Heterocyclic Compounds, 2nd Ed. Interscience Publishers,division of John Wiley & Sons, New York (1967)).

“Substrate” is an entity to which dye compounds of the present inventionare attached. Substrates include, but are not limited to a (i)polynucleotide, (ii) nucleoside and nucleotide, (iii) polypeptide, (iv)carbohydrate, (v) ligand, and (vi) any analog of the preceding (i) to(v).

“Enzymatically incorporatable” is a property of a nucleotide in which itis capable of being enzymatically incorporated onto the terminus, e.g.3′, of a nascent polynucleotide chain through the action of a polymeraseenzyme.

“Terminator” means an enzymatically incorporatable nucleotide whichprevents subsequent incorporations of nucleotides to the resultingpolynucleotide chain and thereby halt polymerase extension. Typicalterminators lack a 3′-hydroxyl substituent and include2′,3′-dideoxyribose, 2′,3′-didehydroribose, and 2′,3′-dideoxy,3′-haloribose, e.g. 3′-fluoro. Alternatively, a ribofuranose analogcould be used, such as arabinose. Exemplary nucleotide terminatorsinclude 2′,3′-dideoxy-β-D-ribofuranosyl, β-D-arabinofuranosyl,3′-deoxy-β-D-arabinofuranosyl, 3′-amino-2′,3′-dideoxy-β-D-ribofuranosyl,and 2′,3′-dideoxy-3′-fluoro-β-D-ribofuranosyl (Chidgeavadze (1984)Nucleic Acids Res., 12: 1671–1686; and Chidgeavadze (1985) FEB. Lett.,183: 275–278). Nucleotide terminators also include reversible nucleotideterminators (Metzker (1994) Nucleic Acids Res., 22(20): 4259).

“Enzymatically extendable” is a property of a nucleotide in which it isenzymatically incorporatable at the terminus of a polynucleotide and theresulting extended polynucleotide can undergo subsequent incorporationsof nucleotides or nucleotide analogs.

V.2 Atropisomer Compounds

The compositions of the invention are asymmetric xanthene compounds thatexist in stable atropisomeric forms. Aryl substituents can restrictrotation around the biannular bond between C-10 and C-15 (noted by anarrow) in the following structure I:

Asymmetric compounds of the invention include xanthene dyescharacterized by the general structure II:

and include asymmetric fluorescent dye classes such as fluorescein (Z¹,Z²=O), rhodol (Z¹=O, Z²=NR₂), and rhodamine (Z¹, Z²=NR₂). Where Z¹ or Z²is NR₂, R may independently be hydrogen, C₁–C₁₂ alkyl, phenyl, benzyl,aryl, heterocycle, or a linking moiety.

Substituents R¹, R⁴, R⁵, R¹¹, R¹³, R¹⁴, R¹⁷, R¹⁸, R¹⁹, and R²⁰ may beindependently fluorine, chlorine, C₁–C₈ alkyl, carboxylate, sulfate,sulfonate (—SO₃ ⁻), alkylsulfonate (—R—SO₃ ⁻), aminomethyl (—CH₂NH₂),aminoalkyl, 4-dialkylaminopyridinium, hydroxymethyl (—CH₂OH), methoxy(—OCH₃), hydroxyalkyl (—ROH), thiomethyl (—CH₂SH), thioalkyl (—RSH),alkylsulfone (—SO₂R), arylthio (—SAr), arylsulfone (—SO₂Ar), sulfonamide(—SO₂NR₂), alkylsulfoxide (—SOR), arylsulfoxide (—SOAr), amino (—NH₂),ammonium (—NH₃ ⁺), amido (—CONR₂), nitrile (—CN), C₁–C₈ alkoxy (—OR),phenoxy, phenolic, tolyl, phenyl, aryl, benzyl, heterocycle,phosphonate, phosphate, quaternary amine, sulfate, polyethyleneoxy, andlinking moiety.

The compounds of the invention include fused benzo rings where R¹³ andR¹⁴, or R⁴ and R⁵, taken together form benzo, and where the fused benzogroups are substituted with substituents.

Substituents R¹, R⁴, R⁵, R¹¹, R¹³, R¹⁴, R¹⁷, R¹⁸, R¹⁹, and R²⁰ may alsobe independently an electron-deficient heterocycle, including 2-pyridyl,3-pyridyl, 4-pyridyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 2-imidazole,4-imidazole, 3-pyrazole, 4-pyrazole, pyridazine, pyrimidine, pyrazine,cinnoline, pthalazine, quinazoline, quinoxaline, 3-(1,2,4-N)-triazolyl,5-(1,2,4-N)-triazolyl, 5-tetrazolyl, 4-(1-O,3-N)-oxazole,5-(1-O,3-N)-oxazole, 4-(1)-S,3-N)-thiazole, 5-(1-S,3-N)-thiazole,2-benzoxazole, 2-benzothiazole, 4-(1,2,3-N)-benzotria or benzimidazole.

Examples of asymmetric fluorescein dyes include the structures:

The compounds of the invention include atropisomeric, asymmetricrhodamines with ring structures formed by the Z¹ nitrogen, the Z¹-bondedcarbon, and the R¹-bonded carbon, to make a first ring structure havingfrom 4 to 7 members. Optionally, the compounds may have a second ringstructure formed by the Z² nitrogen, the Z²-bonded carbon, and theR¹¹-bonded carbon, also having from 4 to 7 members. An example includesthe structure IIa:

Asymmetry results where either: (1) R¹≠R¹¹, R⁴≠R¹⁴, R⁵≠R¹³, or Z¹≠Z²,and (2) R¹⁷≠R¹⁹, or R²⁰≠X. In other words, both aryl substituents on thebiannular sp2-sp2 bond are asymmetric and the compound lacks a C₂ axisof symmetry along the biannular bond axis. C₂ symmetry is defined bytaking the biannular bond in I or II as the axis such that rotation of180° around the axis, gives the same molecule. An example of a symmetricxanthene compound with C₂ symmetry is fluorescein (R¹, R⁴, R⁵, R¹¹, R¹³,R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰═H; X═CO₂H). An example of an asymmetric xanthenecompound without C₂ symmetry is C-11 aminomethyl, C-19carboxyfluorescein (R¹, R⁴, R⁵, R¹³, R¹⁴, R¹⁷, R¹⁸, R²⁰═H; R¹¹═CH₂NH₂;R¹⁹, X═CO₂H). This compound is atropisomeric because the substituentsadjacent to the C-10 to C-15 biannular bond are sufficiently bulky thatrotation is hindered. The energy barrier to rotation is sufficientlyhigh that stable, non-superimposable, mirror image atropisomeric forms2a and 2b result, as shown:

Rotation around the biannular bond results in racemization and loss ofatropisomerism. Typically, racemization of the asymmetric xanthenecompounds occurs upon heating.

The compounds of the present invention can be prepared by any suitablemethod available in the art. Exemplary methods for preparing a varietyof different asymmetric xanthene compounds can be found in the Examplesection below, and as discussed in greater detail below.

As a specific example, reference is made throughout the specification toZ¹ and Z² substituents. As this nomenclature corresponds to theillustrated structural formulae, which represent only one of severalpossible tautomeric forms (or resonance structures) of the compounds, itwill be understood that these references are for convenience only, andthat any such references are not intended to limit the scope of thecompounds described herein.

Those of skill in the art will also recognize that the compounds of theinvention may exist in many different protonation states, depending on,among other things, the pH of their environment. While the structuralformulae provided herein depict the compounds in only one of severalpossible protonation states, it will be understood that these structuresare illustrative only, and that the invention is not limited to anyparticular protonation state—any and all protonated forms of thecompounds are intended to fall within the scope of the invention.

The compounds of the invention may bear multiple positive or negativecharges. The net charge of the dyes of the invention may be eitherpositive or negative. The counter ions associated with the dyes aretypically dictated by the synthesis and/or isolation methods by whichthe compounds are obtained. Typical counter ions include, but are notlimited to ammonium, sodium, potassium, lithium, halides, acetate,trifluoroacetate, etc., and mixtures thereof. It will be understood thatthe identity of any associated counter ion is not a critical feature ofthe invention, and that the invention encompasses the dyes inassociation with any type of counter ion. Moreover, as the compounds canexists in a variety of different forms, the invention is intended toencompass not only forms of the dyes that are in association withcounter ions (e.g., dry salts), but also forms that are not inassociation with counter ions (e.g., aqueous or organic solutions).

Asymmetric xanthene compounds can be conveniently synthesized fromprecursors (U.S. Pat. Nos. 5,188,934; 5,654,442; 5,885,778; 6,096,723;6,020,481; 5,863,727; 5,800,996; 5,945,526; 5,847,162; 6,025,505;6,008,379; 5,936,087; 6,015,719). An exemplary synthetic route starts byaminomethylation of C-19 carboxyfluorescein (Shipchandler (1987) Anal.Biochem. 162:89–101; U.S. Pat. No. 4,510,251; EP 232736; EP110186) togive C-1 (C-11) aminomethyl, C-19 carboxyfluorescein.

An atropisomer substantially free of its stereoisomer may be obtained byresolution of the mixture of stereoisomers of a xanthene compound usinga method such as formation of diastereomers using optically activeresolving agents (“Stereochemistry of Carbon Compounds,” (1962) by E. L.Eliel, McGraw Hill; Lochmuller, C. H., (1975) J. Chromatogr., 113:(3)283–302). Atropisomers of xanthene dyes can be separated and isolated,prior to, or after, derivatization to give reactive labelling reagents.Separation of the atropisomer xanthene compounds of the invention fromthe racemic mixture can be accomplished by any suitable method,including: (1) formation of ionic, diastereomeric salts with chiralcompounds and separation by fractional crystallization or other methods,(2) formation of diastereomeric compounds with chiral derivatizingreagents, separation of the diastereomers, and conversion to the pureatropisomers, and (3) separation of the atropisomers directly underchiral conditions.

Under method (1), diastereomeric salts can be formed by reaction ofenantiomerically pure chiral bases such as brucine, quinine, ephedrine,strychnine, α-methyl-β-phenylethylamine (amphetamine), and the like withasymmetric xanthene compounds bearing acidic functionality, such ascarboxylic acid and sulfonic acid. The diastereomeric salts may beinduced to separate by fractional crystallization or ionicchromatography. For separation of the optical isomers of aminocompounds, addition of chiral carboxylic or sulfonic acids, such ascamphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid canresult in formation of the diastereomeric salts.

Alternatively, by method (2), the substrate to be resolved is reactedwith one enantiomer of a chiral compound to form a diastereomeric pair(Eliel, E. and Wilen, S. (1994) Stereochemistry of Organic Compounds,John Wiley & Sons, Inc., p. 322). Diastereomeric compounds can be formedby reacting asymmetric xanthene compounds with enantiomerically purechiral derivatizing reagents, such as menthyl derivatives, followed byseparation of the diastereomers and hydrolysis to yield the free,enantiomerically enriched xanthene. A method of determining opticalpurity involves making chiral esters, such as a menthyl ester or Mosherester, α-methoxy-α-(trifluoromethyl)phenyl acetate (Jacob III. (1982) J.Org. Chem. 47:4165), of the racemic mixture, and analyzing the NMRspectrum for the presence of the two atropisomeric diastereomers. Forexample, C-1 aminomethyl, C-19 carboxy fluorescein, an asymmetricxanthene compound useful for attaching to nucleotides, polynucleotides,and other fluorescent dyes was reacted with (−) menthyl chloroformate inthe presence of base to form the diastereomeric mixture of menthylcarbamates 1a and 1b (Example 1, FIG. 1 a). Stable diastereomers ofatropisomeric xanthene compounds can be separated and isolated bynormal- and reverse-phase chromatography following methods forseparation of atropisomeric naphthyl-isoquinolines (Hoye, T., WO96/15111). Diastereomers 1a and 1b were separated by preparativereverse-phase HPLC (Example 2, FIG. 1 b).

By method (3), a racemic mixture of two asymmetric enantiomers can beseparated by chromatography using a chiral stationary phase (“ChiralLiquid Chromatography” (1989) W. J. Lough, Ed. Chapman and Hall, NewYork; Okamoto, (1990) “Optical resolution of dihydropyridine enantiomersby High-performance liquid chromatography using phenylcarbamates ofpolysaccharides as a chiral stationary phase”, J. of Chromatogr.513:375–378). Enantiomeric atropisomers of xanthene compounds can beseparated and isolated by chromatography on chiral stationary phase. Asample of racemic, C-1 aminomethyl, C-19 carboxy fluorescein gave twopeaks, resolving the atropisomeric stereoisomers, by HPLC analysis on achiral adsorbent column (FIG. 2 b). When the atropisomers are separated,for example, by the chiral derivatization method (Example 1),preparative HPLC separation (Example 2) and hydrolysis of the chiralmenthyl auxiliaries (Example 3, FIG. 2 a), the separated atropisomer 2ashowed a single peak when analyzed by HPLC on the chiral adsorbentcolumn (FIG. 2 c).

Atropisomers can be distinguished by methods used to distinguish otherchiral molecules with asymmetric carbon atoms, such as optical rotationand circular dichroism.

V.3 Energy-Transfer Dyes

In another aspect, the present invention comprises energy-transfer dyecompounds containing atropisomeric xanthene compounds such as thosedefined by structure II. Generally, the energy-transfer dyes of thepresent invention include a donor dye which absorbs light at a firstwavelength and emits excitation energy in response, an acceptor dyewhich is capable of absorbing the excitation energy emitted by the donordye and fluorescing at a second wavelength in response. The donor dyemay be attached to the acceptor dye through a linker, the linker beingeffective to facilitate efficient energy transfer between the donor andacceptor dyes (Lee, “Energy-transfer dyes with enhanced fluorescence”,U.S. Pat. No. 5,800,996; Lee “Energy-transfer dyes with enhancedfluorescence”, U.S. Pat. No. 5,945,526; Mathies, “Fluorescent labels andtheir use in separations”, U.S. Pat. No. 5,654,419; Lee (1997) NucleicAcids Res. 25:2816–22). Alternatively, the donor dye and the acceptordye may be labelled at different attachment sites on the substrate. Forexample, an oligonucleotide may be labelled with a donor dye at the 5′terminus and an acceptor dye at the 3′ terminus. A polypeptide may belabelled with a donor dye at the carboxyl terminus and an acceptor dyeat an internal cysteine or lysine sidechain (Komoriya, “Compositions forthe detection of proteases in biological samples and methods of usethereof”, U.S. Pat. No. 5,605,809). In the energy-transfer dye of theinvention, at least one of the donor or acceptor dyes which label asubstrate is an atropisomeric xanthene compounds. Other dyes comprisingthe energy-transfer dye may be any fluorescent moiety which undergoesthe energy transfer process with an atropisomeric xanthene compound,including a fluorescein, rhodol, and a rhodamine. Other dyes includeclasses of fluorescent dyes such as cyanine, phthalocyanine, squaraine,bodipy, benzophenoxazine, fluorescein, dibenzorhodamine, or rhodamine.

Energy-transfer dyes have advantages for use in the simultaneousdetection of multiple labelled substrates in a mixture, such as DNAsequencing. A single donor dye can be used in a set of energy-transferdyes so that each dye has strong absorption at a common wavelength. Bythen varying the acceptor dye in the energy-transfer set, the acceptordyes can be spectrally resolved by their respective emission maxima.Energy-transfer dyes also provide a larger effective Stokes shift thannon-energy-transfer dyes. The Stokes shift is the difference between theexcitation maximum, the wavelength at which the donor dye maximallyabsorbs light, and the emission maximum, the wavelength at which theacceptor maximally emits light.

Generally the linker between the donor dye and acceptor dye has thestructures:

wherein Z is selected from the group consisting of NH, S and O; R²¹ is aC₁–C₁₂ alkyl attached to the donor dye; R²² is a substituent selectedfrom the group consisting of a C₁–C₁₂ alkyldiyl, a five and six memberedring having at least one unsaturated bond and a fused ring structurewhich is attached to the carbonyl carbon; and R²³ includes a functionalgroup which attaches the linker to the acceptor dye. R²² may be a fiveor six membered ring such as cyclopentene, cyclohexene, cyclopentadiene,cyclohexadiene, furan, thiofuran, pyrrole, isopyrole, isoazole,pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine,pyrimidine, pyrazine oxazine, indene, benzofuran, thionaphthene, indoleand naphthalene. Specifically, the linker may have the structure:

where n ranges from 2 to 10.

Generally also, R²³ may have the structure:

wherein R²⁴ is a C₁–C₁₂ alkyl and

In one embodiment, the linker between the donor dye and acceptor dyeincludes a functional group which gives the linker some degree ofstructural rigidity, such as an alkene, diene, an alkyne, a five and sixmembered ring having at least one unsaturated bond or a fused ringstructure. The donor dye and the acceptor dye of the energy-transfer dyemay be attached by linkers which have the exemplary structures:

wherein D is a donor dye, A is an acceptor dye and n is 1 or 2. Thephenyl rings may be substituted with groups such as sulfonate,phosphonate, and other charged groups.

The attachment sites of the linker between the donor dye and acceptordye of an energy-transfer dye may be at any position where one or bothof the donor dye and acceptor dye is a compound of the presentinvention. Exemplary attachment sites include R¹, R¹¹, R¹⁸, R¹⁹, Z¹ andZ². Examples of linkers and attachment sites are shown in terminatornucleotides 25, 26, 33, and 34 where the linkers attach at R¹ or R¹¹ ofthe donor fluorescein dye and at R¹⁸ or R¹⁹ of the acceptor rhodaminedye. An alternative embodiment is where the donor dye and the acceptordye are attached by a linker through the R¹⁸ or R¹⁹ sites, and eitherthe donor dye or the acceptor dye is attached to a substrate through theR¹, R¹¹, Z¹ or Z² site. Another alternative embodiment is where thedonor dye and the acceptor dye are attached by a linker through the R¹,R¹¹, Z¹ or Z² sites, and either the donor dye or the acceptor dye isattached to a substrate through the R¹⁸ or R¹⁹ site.

The energy-transfer dye compound is covalently attached to a substratethrough a linker. The linker may be a bond, C₁–C₁₂ alkyldiyl or C₆–C₂₀aryldiyl. The linker may bear functional groups including amide,carbamate, urea, thiourea, phosphate, phosphonate, sulfonate,phosphorothioate, and the like. Preferred linkers include 1,2-ethyldiyland 1,6-hexyldiyl. The attachment sites of the linker between theenergy-transfer dye and the substrate may be at any position on theenergy-transfer dye, where one or both of the donor dye and acceptor dyeis a dye of the present invention. Where the substrate is a nucleosideor nucleotide, a preferred attachment site on the substrate is on thenucleobase. If the nucleobase is a purine, the linker may be attached atthe 8-position. If the nucleobase is a 7-deazapurine, the linker may beattached at the 7-position or 8-position. If the nucleobase is apyrimidine, the linker may be attached at the 5-position. As examples,in terminator nucleotide examples 25 and 26, the energy-transfer dye isattached to the nucleobase at R¹⁹. Where the substrate is anoligonucleotide, preferred attachment sites include the 3′ and 5′terminii. Other oligonucleotide attachment sites include theinternucleotide phosphate, or phosphate-analog linkage, or at a positionon the sugar, e.g. 2′ or 4′. Where the substrate is a polypeptide(peptide or protein), preferred attachment sites include the amino andcarboxyl termini, and lysine residue amino substituents.

V.4 Methods of Labelling

The present invention comprises labelling reagents wherein atropisomericxanthene compounds are in reactive form to react with substrates. Inanother aspect, the present invention comprises substrates labelled,i.e. conjugated, with the compounds of the invention, formula I.Substrates can be virtually any molecule or substance to which the dyesof the invention can be conjugated, including by way of example and notlimitation, a polynucleotide, a nucleotide, a nucleoside, a polypeptide,a carbohydrate, a ligand, a substantially enantiomerically purecompound, a particle, a surface, a lipid, a solid support, organic andinorganic polymers, and combinations and assemblages thereof, such aschromosomes, nuclei, living cells (e.g., bacteria or othermicroorganisms, mammalian cells, tissues, etc.), and the like. Aparticle may include a nanoparticle, a microsphere, a bead, or aliposome. A surface may be glass or other non-porous planar material.The compounds of the invention are conjugated with the substrate via anoptional linker by a variety of means, including hydrophobic attraction,ionic attraction, and covalent attachment.

Labelling typically results from mixing an appropriate reactiveatropisomeric xanthene and a substrate to be conjugated in a suitablesolvent, using methods well-known in the art (Hermanson, BioconjugateTechniques, (1996) Academic Press, San Diego, Calif. pp. 40–55, 643–71),followed by separation of the labelled substrate, conjugate, from anyunconjugated starting materials or unwanted by-products. The conjugatecan be stored dry or in solution for later use.

A racemic mixture of atropisomeric xanthenes may be separated to isolatesubstantially pure atropisomers at any intermediate stage in thesynthesis of the labelling reagents, according to the aforementionedseparation and isolation methods (1), (2), and (3).

The atropisomeric xanthene may include a linking moiety at one of thesubstituent positions or covalent attachment of the dye to anothermolecule. A linking moiety is typically an electrophilic functionalgroup, capable of forming a covalent bond by reacting with nucleophilicfunctionality on a substrate. Nucleophilic functionality may include,for example, alcohols, alkoxides, amines, hydroxylamines, and thiols.Alternatively, a linking moiety may include nucleophilic functionalitythat reacts with an electrophilic group on a substrate. Examples oflinking moieties include azido, monosubstituted primary amine,disubstituted secondary amine, thiol, hydroxyl, halide, epoxide,N-hydroxysuccinimidyl ester, carboxyl, isothiocyanate, sulfonylchloride, sulfonate ester, silyl halide, chlorotriazinyl, succinimidylester, pentafluorophenyl ester, maleimide, haloacetyl, epoxide,alkylhalide, allyl halide, aldehyde, ketone, acylazide, anhydride,iodoacetamide and an activated ester.

One linking moiety is N-hydroxysuccinimidyl ester (NHS) of a carboxylgroup substituent of the atropisomeric xanthene compound (FIGS. 3, 6,10, 11). The NHS ester form of the compound is a labelling reagent. TheNHS ester of the dye may be preformed, isolated, purified, and/orcharacterized, or it may be formed in situ and reacted with anucleophilic group of a substrate, such as an oligonucleotide, anucleotide, a polypeptide, or the like (Brinkley, M. (1992) BioconjugateChem. 3:2–13). Typically, the carboxyl form of the dye is activated byreacting with some combination of: (1) a carbodiimide reagent, e.g.dicyclohexylcarbodiimide, diisopropylcarbodiimide, or a uronium reagent,e.g. TSTU (O—(N-Succinimidyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate, HBTU(O-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate),HATU (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate); (2) an activator, such as 1-hydroxybenzotriazole(HOBt); and (3) N-hydroxysuccinimide to give the NHS ester of the dye. Arepresentative example of an NHS ester are structures 4a and 4b (FIG.3), 8 (FIG. 4), and 13 (FIG. 5).

Functional groups on an atropisomeric xanthene compound may be protectedprior to derivatization and reaction at other functional groups on thecompound. For example, the amino group of atropisomers 2a and 2b weretrifluoroacetylated to give 3a and 3b, separately (FIG. 2 a, Examples 5and 6). The carboxyl groups of were then converted to the active ester,NHS with N-hydroxysuccinimide and a carbodiimide reagent, e.g. DAE togive 4a and 4b, separately (FIG. 3, Examples 7 and 8).

In some cases, the atropisomeric xanthene compound and the substrate maybe coupled by in situ activation of the compound and reaction with thesubstrate to form the atropisomeric xanthene-substrate conjugate in onestep. Other activating and coupling reagents include TBTU(2-(1H-benzotriazo-1-yl)-1-1,3,3-tetramethyluroniumhexafluorophosphate), TFFH(N,N′,N″,N′″-tetramethyluronium2-fluoro-hexafluorophosphate), PyBOP(benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate, EEDQ(2-ethoxy-1-ethoxycarbonyl-1,2-dihydro-quinoline), DCC(dicyclohexylcarbodiimide); DIPCDI (diisopropylcarbodiimide), MSNT(1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole, and aryl sulfonylhalides, e.g. triisopropylbenzenesulfonyl chloride.

Another preferred reactive linking group is a phosphoramidite form ofasymmetric xanthene compounds. Phosphoramidite dye reagents areparticularly useful for the automated synthesis of oligonucleotideslabelled with the dyes of the invention. Most conveniently,phosphoramidite dye reagents may be coupled to oligonucleotides bound toa solid support during the normal course of solid-phase synthesis.Oligonucleotides are commonly synthesized on solid supports by thephosphoramidite method (Caruthers, M. and Beaucage, S. “Phosphoramiditecompounds and processes”, U.S. Pat. No. 4,415,732; Caruthers, M. andMatteucci, M. “Process for preparing polynucleotides”, U.S. Pat. No.4,458,066; Beaucage, S. and Iyer, R. (1992) “Advances in the synthesisof oligonucleotides by the phosphoramidite approach”, Tetrahedron48:2223–2311).

Phosphoramidite atropisomeric xanthene reagents can be nucleosidic ornon-nucleosidic. Non-nucleosidic forms of the phosphoramidite reagentshave the general formula III:

where DYE is a protected or unprotected form of atropisomer xanthene II,including energy-transfer dye; L is a linker; R³⁰ and R³¹ takenseparately are C₁–C₁₂ alkyl, C₄–C₁₀ aryl, and cycloalkyl containing upto 10 carbon atoms, or R³⁰ and R³¹ taken together with thephosphoramidite nitrogen atom form a saturated nitrogen heterocycle; andR³² is a phosphite ester protecting group which prevents unwantedextension of the oligonucleotide. Generally, R³² is stable tooligonucleotide synthesis conditions yet is able to be removed from asynthetic oligonucleotide product with a reagent that does not adverselyaffect the integrity of the oligonucleotide or the dye. R³² may be: (i)methyl, (ii) 2-cyanoethyl; —CH₂CH₂CN, or (iii) 2-(4-nitrophenyl)ethyl;—CH₂CH₂(p-NO₂Ph). Embodiments of phosphoramidite reagents include where:(i) R³⁰ and R³¹ are each isopropyl, (ii) R³⁰ and R³¹ taken together ismorpholino, (iii) L is C₁–C₁₂ alkyl, (iv) R³² is 2-cyanoethyl, and (v)DYE is attached at R¹⁸ or R¹⁹ by a linker. The linker, L, mayalternatively be:

where n ranges from 1 to 10. An example of phosphoramidite reagent IIIhas the structure:

Phosphoramidite dye reagents III effect labelling of a substrate with asingle, substantially pure, atropisomeric xanthene of the invention.Where the substrate is an oligonucleotide, the dye will be attached atthe 5′ terminus of the oligonucleotide, as a consequence of the typical3′ to 5′ direction of synthesis, or at the 3′ terminus of theoligonucleotide when the 5′ to 3′ direction synthesis method ispracticed (Wagner (1997) Nucleosides & Nucleotides 16:1657–60). ReagentIII may be coupled to a polynucleotide which is bound to a solidsupport, e.g. through the 3′ terminus. Other phosphoramidite dyereagents, nucleosidic and non-nucleosidic allow for labelling at othersites of an oligonucleotide, e.g. 3′ terminus, nucleobase,internucleotide linkage, sugar. Labelling at the nucleobase,internucleotide linkage, and sugar sites allows for internal andmultiple labelling with fluorescent dyes.

An atropisomeric xanthene compound of the invention may be converted toa non-nucleosidic, phosphoramidite labelling reagent by any known methodof phosphitylation of nucleophilic functionality with trivalentphosphitylating reagents. For example, when the xanthene contains acarboxyl group, e.g. R¹⁹═CO₂H, the carboxyl may be activated, e.g. tothe NHS, and amidated with 6-amino-1-hexanol. The resulting hydroxyl maybe phosphitylated with bis(diisopropylamino)cyanoethylphosphite orchloro-diisopropylamino-cyanoethylphosphine to give the phosphoramiditedye-labelling reagent (Theisen (1992) “Fluorescent dye phosphoramiditelabelling of oligonucleotides”, in Nucleic Acid Symposium Series No. 27,Oxford University Press, Oxford, pp. 99–100). Alternatively, thecarboxyl group of the compound may be reduced to the hydroxyl, to bephosphitylated.

The phosphoramidite reagent III reacts with a hydroxyl group, e.g. 5′terminal OH of an oligonucleotide bound to a solid support, under mildacid activation, to form an internucleotide phosphite group which isthen oxidized to an internucleotide phosphate group. In some instances,the xanthene compound may contain functional groups, e.g. Z¹ and Z²oxygens as in structure I, that require protection either during thesynthesis of the phosphoramidite reagent or during its subsequent use tolabel molecules such as oligonucleotides. The protecting group(s) usedwill depend upon the nature of the functional groups, and will beapparent to those having skill in the art (Greene, T. and Wuts, P.Protective Groups in Organic Synthesis, 2nd Ed., John Wiley & Sons, NewYork, 1991). Generally, the protecting groups used should be stableunder the acidic conditions (e.g. trichloroacetic acid, dichloroaceticacid) commonly employed in oligonucleotide synthesis to remove5′-hydroxyl protecting groups (e.g., dimethoxytrityl) and labile underthe basic conditions (ammonium hydroxide, aqueous methylamine) used todeprotect and/or cleave synthetic oligonucleotides from solid supports.

Polypeptides, antibodies, and other biopolymers comprised of amino acidsand amino acid analogs may be covalently labelled by conjugation withthe atropisomeric xanthene compounds of the invention. Typically, thecompound is in electrophilic form, e.g. NHS reactive linking group,which reacts with a nucleophilic group of the peptide, e.g. aminoterminus, or amino side chain of an amino acid such as lysine.Alternatively, the dye may be in nucleophilic form, e.g. amino- orthiol-reactive linking group, which may react with an electrophilicgroup of the peptide, e.g. NHS of the carboxyl terminus or carboxyl sidechain of an amino acid. Labelled polypeptides may retain their specificbinding and recognition properties in interacting with cell surface andintracellular components. The xanthene compound, acting as a dye,provides a detection element for localizing, visualizing, andquantitating the binding or recognition event. Polypeptides can also belabelled with two moieties, a fluorescent reporter and quencher, whichtogether undergo fluorescence resonance energy transfer (FRET). Thefluorescent reporter may be partially or significantly quenched by thequencher moiety in an intact polypeptide. Upon cleavage of thepolypeptide by a peptidase or protease, a detectable increase influorescence may be measured (Knight, C. (1995) “Fluorimetric Assays ofProteolytic Enzymes”, Methods in Enzymology, Academic Press, 248:18–34).

V.4A Labelled Nucleotides

A preferred class of labelled substrates include conjugates ofnucleosides and nucleotides that are labelled with the dyes of theinvention. Such labelled nucleosides and nucleotides are particularlyuseful for labelling polynucleotides formed by enzymatic synthesis,e.g., labelled nucleotide 5′-triphosphates used in the context of PCRamplification, Sanger-type polynucleotide sequencing, andnick-translation reactions.

Nucleosides and nucleotides can be labelled at sites on the sugar ornucleobase moieties. Preferred nucleobase labelling sites include the8-C of a purine nucleobase, the 7-C or 8-C of a 7-deazapurinenucleobase, and the 5-position of a pyrimidine nucleobase. Between anucleoside or nucleotide and a dye, a linker may attach to anatropisomeric xanthene compound at any position.

The labelled nucleoside or nucleotide may be enzymaticallyincorporatable and enzymatically extendable. Nucleosides or nucleotideslabelled with compounds of the invention may have formula IV:

where DYE is a protected or unprotected form of compounds I or II,including energy-transfer dye. B may be any nucleobase, e.g. uracil,thymine, cytosine, adenine, 7-deazaadenine, guanine, and8-deazaguanosine. R²⁵ is H, monophosphate, diphosphate, triphosphate,thiophosphate, or phosphate ester analog. R²⁶ and R²⁷, when taken alone,are each independently H, HO, F and a phosphoramidite. Where R²⁶ or R²⁷is phosphoramidite, R²⁵ is an acid-cleavable hydroxylprotecting group,e.g. dimethoxytrityl, which allows subsequent monomer coupling underautomated synthesis conditions (Caruthers, “Phosphoramidite compoundsand processes”, U.S. Pat. No. 4,415,732; Caruthers, “Process forpreparing polynucleotides”, U.S. Pat. No. 4,458,066; Beaucage, S. andIyer, R. (1992) “Advances in the synthesis of oligonucleotides by thephosphoramidite approach”, Tetrahedron 48:2223–2311).

Where the labelled nucleoside or nucleotide is a terminator, R²⁶ and R²⁷are selected to block polymerase-mediated template-directedpolymerization. In terminator nucleotides, R²⁶ and R²⁷, when takenalone, are each independently H, F, and a moiety which blockspolymerase-mediated template-directed polymerization, or when takentogether form 2′-3′-didehydroribose. In formula IV, when both R²⁶ andR²⁷ are hydroxyl, the resultant compounds are labelled ribonucleosidesand ribonucleotides (NTP). When R²⁷ is hydrogen and R²⁶ is hydroxyl, theresultant compounds are labelled 2′-deoxyribonucleosides and nucleotides(dNTP). When R²⁶ and R²⁷ are each hydrogen, the resultant compounds are2′,3′-dideoxyribonucleosides and nucleotides (ddNTP). Labelled ddNTPfind particular use as terminators in Sanger-type DNA sequencing methodsutilizing fluorescent detection. Labelled2′-deoxyribonucleoside-5′-triphosphates (dNTP) find particular use asreagents for labelling DNA polymerase extension products, e.g., in thepolymerase chain reaction or nick-translation. Labelledribonucleoside-5′-triphosphates (NTP) find particular use as reagentsfor labelling RNA polymerase extension products.

Alkynylamino-linked compounds IV, where L includes an alkyndiyl group,are useful for conjugating atropisomeric xanthene compounds tonucleosides, nucleotides and analogs therein. Their synthesis is taughtin EP 87305844.0 and Hobbs, (1989) J. Org. Chem. 54:3420. Thecorresponding nucleoside mono-, di- and triphosphates are obtained bystandard techniques (for example, the methods described in U.S. Pat.Nos. 5,821,356; 5,770,716; 5,948,648; 6,096,875). Methods forsynthesizing compounds IV with modified propargylethoxyamido linkers Lcan also be found in these patents. Additional synthesis proceduressuitable for use in synthesizing compounds according to structuralformula IV are described, for example, in Gibson (1987) Nucl. Acids Res.15:6455–6467; Gebeyehu (1987) Nucl. Acids Res. 15:4513–4535;Haralambidis (1987) Nucl. Acids Res. 15:4856–4876; Nelson (1986)Nucleosides and Nucleotides. 5(3):233–241; Bergstrom (1989) J. Am. Chem.Soc. 111:374–375; U.S. Pat. No. 4,855,225, U.S. Pat. No. 5,231,191 andU.S. Pat. No. 5,449,767, which are incorporated herein by reference. Anyof these methods can be routinely adapted or modified as necessary tosynthesize the full range of labelled nucleosides, nucleotides, andanalogs described herein.

One embodiment of the alkynyl linker L may be:

wherein n is 0, 1, or 2.

Energy-transfer dye pairs can be conjugated to a nucleotide5′-triphosphate by linking through a nucleobase amino group to: (i) anactivated ester of a energy-transfer dye pair, or (ii) stepwise couplingto one dye, e.g. R¹¹-protected aminomethyl, R¹⁸-carboxyl fluorescein,then coupling the unprotected R¹¹-aminomethyl to the second dye of thepair.

Linker reagents may be prepared by known synthetic methods. For example,phosphate linker reagent 5 is synthesized starting from the cyclicphosphoramidite 7. Phosphitylation of 7 with methyl glycolate 6 wasfollowed by in situ oxidation to the pentavalent phosphate 8. Hydrolysisof the methyl ester, the trifluoroacetate group, and demethylation gave9. Protection of the amino group with Fmoc gave 10 which was activatedas the N-hydroxysuccinimide ester, linker reagent 5 (FIG. 4, Example 9).

An alkynylamino-linked nucleotide can be prepared by first coupling NHSlinker reagent 5 with 7-deaza-7-propargylamino-ddATP 12 to give 13,followed by hydrolysis of the Fmoc group to give 11 (FIG. 5, Example10). The amino atropisomeric xanthene 1a is coupled with the N-Fmoc, NHSester of p-aminomethylbenzoic acid (Example 11) and then activated asthe NHS ester to give 14 (FIG. 6). Reaction of 11 and 14 gave theatropisomeric xanthene ddATP compound 16. The Fmoc group was removedwith ammonium hydroxide to give 15 (FIG. 6, Example 12).

The NHS-rhodamine dye 17 was synthesized from bicyclic amine 18.Cyclization with 1-bromo-3-chloropropane gave tricyclic ester 19, whichwas hydrolyzed to tricyclic amine 20 (FIG. 7, Example 13).Friedel-Crafts acylation of 20 with anhydride 21 gave the ketone 22which was reacted with another equivalent of to give symmetric rhodamineisopropyl ester 23. The ester of 23 was cleaved and the carboxylic acid24 was converted to NHS-rhodamine dye 17 (FIG. 8, Example 13).

The substantially pure atropisomer xanthene energy transfer ddATPterminator 25 was formed by coupling 15 with 17, followed byanion-exchange HPLC purification (FIG. 9, Example 14).

Alternative synthetic routes to energy-transfer nucleotides andpolynucleotides, with different convergent schemes may be practiced. Thesubstrate, dye, and linker subunits, or synthons, may be assembled forcoupling in any order. For example, the energy-transfer pair of donordye and acceptor dye may be covalently attached through a linker andthen coupled to the nucleotide or polynucleotide. Many differentsynthetic routes can be practiced which result in the labelling ofnucleotides with the dyes of the invention. Reactive functionality, suchas carboxylic acid, amino, hydroxyl groups, may require protection,utilizing the vast art of organic synthesis methodology.

Another rhodamine dye 28 was protected as the bis-trifluoroacetamide 29and converted to the NHS compound 27 (FIG. 10, Example 15).Propargylethoxyamino ddTTP 30 was coupled with atropisomer xanthenecompound 14 to give Fmoc atropisomer xanthene ddTTP 31 which washydrolyzed to 32 (FIG. 11, Example 16). Reaction of 27 and 32 gaveatropisomer, energy-transfer terminator ddTTP 26, purified byanion-exchange HPLC (FIG. 12, Example 17).

V.4B Labelled Oligonucleotides

Oligonucleotides are commonly synthesized on solid supports by thephosphoramidite method (U.S. Pat. Nos. 4,415,732; 4,973,679; 4,458,066;Beaucage, S. and Iyer, R. (1992) Tetrahedron 48:2223–2311) usingcommercially available phosphoramidite nucleosides, supports e.g.silica, controlled-pore-glass (U.S. Pat. No. 4,458,066) and polystyrene(U.S. Pat. Nos. 5,047,524 and 5,262,530) and automated synthesizers(Models 392, 394, 3948 DNA/RNA Synthesizers, Applied Biosystems).

Another preferred class of labelled substrates include conjugates ofoligonucleotides and the compounds of the invention. Such conjugates mayfind utility as DNA sequencing primers, PCR primers, oligonucleotidehybridization probes, oligonucleotide ligation probes, double-labelled5′-exonuclease (TaqMan™) probes, and the like (Fung, U.S. Pat. No.4,757,141; Andrus, “Chemical methods for 5′ non-isotopic labelling ofPCR probes and primers” (1995) in PCR 2: A Practical Approach, OxfordUniversity Press, Oxford, pp. 39–54; Hermanson, Bioconjugate Techniques,(1996) Academic Press, San Diego, Calif. pp. 40–55, 643–71; Mullah(1998) “Efficient synthesis of double dye-labelledoligodeoxyribonucleotide probes and their application in a real time PCRassay”, Nucl. Acids Res. 26:1026–1031). A labelled oligonucleotide mayhave formula V:

where the oligonucleotide comprises 2 to 100 nucleotides. DYE is aprotected or unprotected form of compounds I or II, includingenergy-transfer dye. B is any nucleobase, e.g. uracil, thymine,cytosine, adenine, 7-deazaadenine, guanine, and 8-deazaguanosine. L is alinker. R²⁷ is H, OH, halide, azide, amine, C₁–C₆ aminoalkyl, C₁–C₆alkyl, allyl, C₁–C₆ alkoxy, OCH₃, or OCH₂CH═CH₂. R²² is H, phosphate,internucleotide phosphodiester, or internucleotide analog. R²⁹ is H,phosphate, internucleotide phosphodiester, or internucleotide analog. Inthis embodiment, structure V, the nucleobase-labelled oligonucleotidemay bear multiple dyes of the invention attached through thenucleobases. Nucleobase-labelled oligonucleotide V may be formed by: (i)enzymatic incorporation of enzymatically incorporatable nucleotidereagents IV where R²⁵ is triphosphate, by a DNA polymerase or ligase,and (ii) coupling of a nucleoside phosphoramidite reagent by automatedsynthesis. Whereas, nucleobase-labelled oligonucleotides V may bemultiply labelled by incorporation of more than one incorporatablenucleotide IV, labelling with a dye label reagent such as III leads tosingly 5′-labelled oligonucleotides, according to formula VI:

where X is O, NH, or S; R²⁷ is H, OH, halide, azide, amine, C₁–C₆aminoalkyl, C₁–C₆ alkyl, allyl, C₁–C₆ alkoxy, OCH₃, or OCH₂CH═CH₂; R²⁸is H, phosphate, internucleotide phosphodiester, or internucleotideanalog; and L is C₁–C₁₂ alkyl, aryl, or polyethyleneoxy of up to 100ethyleneoxy units.

The linker L in formulas V or VI may be attached at any site on theatropisomeric xanthene compound of the invention, DYE, including R¹,R¹¹, R¹⁸, R¹⁹, Z¹ and Z² of structure I.

In a first method for labelling synthetic oligonucleotides, anucleophilic functionality, e.g. a primary aliphatic amine, isintroduced at a labelling attachment site on an oligonucleotide, e.g. a5′ terminus. After automated, solid-support synthesis is complete, theoligonucleotide is cleaved from the support and all protecting groupsare removed. The nucleophile-oligonucleotide is reacted with an excessof a label reagent containing an electrophilic moiety, e.g.isothiocyanate or activated ester, e.g. N-hydroxysuccinimide (NHS),under homogeneous solution conditions (Hermanson, BioconjugateTechniques, (1996) Academic Press, San Diego, Calif. pp. 40–55, 643–71;Andrus, A. “Chemical methods for 5′ non-isotopic labelling of PCR probesand primers” (1995) in PCR 2: A Practical Approach, Oxford UniversityPress, Oxford, pp. 39–54). Labelled oligonucleotides VI may be formed byreacting a reactive linking group form, e.g. NHS, of a dye, with a5′-aminoalkyl oligonucleotide.

In a second method, a label is directly incorporated into theoligonucleotide during or prior to automated synthesis, for example as asupport reagent (Mullah, “Solid support reagents for the directsynthesis of 3′-labelled polynucleotides”, U.S. Pat. No. 5,736,626;Nelson, “Multifunctional controlled pore glass reagent for solid phaseoligonucleotide synthesis”, U.S. Pat. No. 5,141,813) or as aphosphoramidite reagent III. Certain fluorescent dyes and other labelshave been functionalized as phosphoramidite reagents for 5′ labelling(Theisen (1992) Nucleic Acid Symposium Series No. 27, Oxford UniversityPress, Oxford, pp. 99–100).

Generally, if the labelled oligonucleotide is made by enzymaticsynthesis, the following procedure may be used. A target DNA isdenatured and an oligonucleotide primer is annealed to the template DNA.A mixture of enzymatically-incorporatable nucleotides or nucleotideanalogs capable of supporting continuous template-directed enzymaticextension of the primed target (e.g., a mixture including dGTP, dATP,dCTP and dTTP or dUTP) is added to the primed target. At least afraction of the nucleotides are labelled terminators IV, labelled withan atropisomer xanthene dye II. A polymerase enzyme is next added to themixture under conditions where the polymerase enzyme is active. Alabelled oligonucleotide is formed by the incorporation of the labellednucleotides or terminators during polymerase-mediated strand synthesis.In an alternative enzymatic synthesis method, two primers are usedinstead of one: one complementary to the (+) strand of the target andanother complementary to the (−) strand of the target, the polymerase isa thermostable polymerase and the reaction temperature is cycled betweena denaturation temperature and an extension temperature, therebyexponentially synthesizing a labelled complement to the target sequenceby PCR (Innis (1990) PCR Protocols, Eds., Academic Press).

In one preferred post-synthesis chemical labelling method anoligonucleotide is labelled as follows. An NHS form of a dye accordingto structure I is dissolved or suspended in DMSO and added in excess(5–20 equivalents) to a 5′-aminohexyl oligonucleotide in 0.25 Mbicarbonate/carbonate buffer at about pH 9 and allowed to react for 6hours, e.g., U.S. Pat. No. 4,757,141. The atropisomer xanthene labelledoligonucleotide can be separated from unreacted dye by passage through asize-exclusion chromatography column eluting with buffer, e.g., 0.1molar triethylamine acetate (TEAA). The fraction containing the crudelabelled oligonucleotide is further purified by reverse phase HPLCemploying gradient elution.

Polynucleotides labelled with the atropisomer xanthene compounds of thepresent invention may be additionally labelled with moieties that affectthe rate of electrophoretic migration, i.e. mobility-modifying labels.Mobility-modifying labels include polyethyleneoxy units, —(CH₂CH₂O)_(n)—where n may be 1 to 100 (Grossman, U.S. Pat. No. 5,624,800). Preferably,n is from 2 to 20. The polyethyleneoxy units may be interspersed withphosphate groups. Specifically labelling atropisomer xanthene-labelledpolynucleotides with additional labels of polyethyleneoxy of discreteand known size allows for separation by electrophoresis, substantiallyindependent of the number of nucleotides in the polynucleotide. That is,polynucleotides of the same length may be discriminated upon by thepresence of spectrally resolvable dye labels and mobility-modifyinglabels. Polynucleotides bearing both dye labels and mobility-modifyinglabels may be formed enzymatically by ligation or polymerase extensionof the single-labelled polynucleotide or nucleotide constituents.

V.5 Methods

Methods requiring simultaneous detection of multiplespatially-overlapping analytes may benefit from substantially pureatropisomers of asymmetric xanthene dyes as labels. The atropisomerxanthene compounds of the present invention are well suited for anymethod utilizing fluorescent detection, such as polymerase chainreaction (PCR) amplification, DNA sequencing, antisense transcriptionaland translational control of gene expression, genetic analysis, and DNAprobe-based diagnostic testing (Kricka, L. (1992) Nonisotopic DNA ProbeTechniques, Academic Press, San Diego, pp. 3–28). Fluorescence detectionof fluorescent dye-labelled oligonucleotides is the basis for nucleicacid sequence detection assays such as 5′ exonuclease assay (Livak, U.S.Pat. No. 5,723,591), FRET hybridization (Tyagi, S. and Kramer, F. (1996)“Molecular Beacons: Probes that fluoresce upon hybridization”, NatureBiotechnology, 14:303–08), genetic linkage mapping (Dib (1996) “Acomprehensive genetic map of the human genome based on 5,264microsatellites”, Nature 380:152–54) and oligonucleotide-ligation assay(Grossman (1994) “High-density multiplex detection of nucleic acidsequences: oligonucleotide ligation assay and sequence-codedseparation”, Nucl. Acids Res. 22:4527–34).

The present invention is particularly well suited for detecting classesof differently-labelled polynucleotides that have been subjected to abiochemical separation procedure, such as electrophoresis (Rickwood andHames, Eds., Gel Electrophoresis of Nucleic Acids: A Practical Approach,IRL Press Limited, London, 1981). The electrophoretic matrix may be asieving polymer, e.g. crosslinked or uncrosslinked polyacrylamide, orother amide-containing polymer, having a concentration (weight tovolume) of between about 2–20 weight percent (Madabhushi, U.S. Pat. Nos.5,552,028; 5,567,292; 5,916,426). The electrophoretic matrix may beconfigured in a slab gel or capillary format (Rosenblum, (1997) NucleicAcids Res. 25:3925–29; Mathies, U.S. Pat. No. 5,274,240).

V.5A Primer Extension

In a preferred category of methods referred to herein as “fragmentanalysis” or “genetic analysis” methods, polynucleotide fragmentslabelled with fluorescent dyes, including substantially pureatropisomeric xanthene compounds, are generated throughtemplate-directed enzymatic synthesis using labelled primers ornucleotides, e.g. by ligation or polymerase-directed primer extension.The polynucleotide fragments may be subjected to a size-dependentseparation process, e.g., electrophoresis or chromatography, and theseparated fragments are detected subsequent to the separation, e.g., bylaser-induced fluorescence (Hunkapiller, U.S. Pat. No. 4,811,218).Multiple classes of polynucleotides may be separated simultaneously andthe different classes are distinguished by spectrally resolvable labels,including dyes of the invention. In electrophoresis, the classesseparate on the basis of electrophoretic migration rate.

V.5B DNA Sequencing

Preferably, the chain termination methods of DNA sequencing, i.e.dideoxy DNA sequencing, or Sanger-type sequencing, and fragment analysisis employed (Sanger (1977) “DNA sequencing with chain-terminatinginhibitors”, Proc. Natl. Acad. Sci. USA 74:5463–5467). Exemplarychain-terminating nucleotide analogs include the 2′,3′-dideoxynucleoside5′-triphosphates (ddNTP) which lack the 3′-OH group necessary for 3′ to5′ DNA chain elongation. Primers or ddNTP may be labelled with thesubstantially pure atropisomer xanthene dyes of the invention anddetected by fluorescence after separation of the fragments byhigh-resolution electrophoresis. Dyes can be linked to functionality onthe 5′ terminus of the primer, e.g. amino (Fung, U.S. Pat. No.4,757,141), on the nucleobase of a primer; or on the nucleobase of adideoxynucleotide, e.g. via alkynylamino linking groups (Khan, U.S. Pat.Nos. 5,770,716; and 5,821,356; Hobbs, U.S. Pat. No. 5,151,507).

Each of the terminators bears a different fluorescent dye andcollectively the terminators of the experiment bear a set of dyesincluding one or more from the dyes of the invention. In a preferredfragment analysis method, fragments labelled with dyes are identified byrelative size, i.e. sequence length. Correspondence between fragmentsize and sequence is established by incorporation of the four possibleterminating nucleotides (“terminators”) and the members of a set ofspectrally resolvable dyes (Bergot, U.S. Pat. No. 5,366,860). The set ofspectrally resolvable dyes may include at least one substantially pureatropisomeric xanthene compound.

V.6 Ligation

The covalent joining of nucleic acid probes by ligase enzymes is one ofthe most useful tools available to molecular biologists. When two probesare annealed to a template nucleic acid where the two probes areadjacent and without intervening gaps, a phosphodiester bond can beformed between a 5′ terminus of one probe and the 3′ terminus of theother probe by a ligase enzyme, (Whiteley, U.S. Pat. No. 4,883,750;Landegren, (1988) “A ligase mediated gene detection technique”, Science241:1077–80; Nickerson, “Automated DNA diagnostics using an ELISA-basedoligonucleotide assay” (1990) Proc. Natl. Acad. Sci USA 87:8923–27).Oligonucleotide ligation assays detect the presence of specificsequences in target DNA sample. Where one or both probes are labelledwith a dye, the ligation product may be detected by fluorescence. One orboth probes may be labelled with a substantially pure atropisomericxanthene dye. Ligation products may be detected by electrophoresis,chromatography, or other size- or charge-based separation method.

V.7 Amplification

The atropisomer xanthene compounds of the invention find applications aslabels on 5′-labelled oligonucleotide primers for the polymerase chainreaction (PCR) and other nucleic acid amplification and selectionmethods. PCR applications include the use of labelled oligonucleotidesfor genotyping by variable number tandem repeat (VNTR), short tandemrepeat (STR), and microsatellite methods of amplification of repeatregions of double-stranded DNA that contain adjacent multiple copies ofa particular sequence, with the number of repeating units beingvariable. Preferably, in such PCR genotyping methods, the PCR primer islabelled with an atropisomer xanthene of the invention.

In a particularly preferred embodiment, atropisomer xanthene compoundsmay be used in quantitative methods and reagents that provide real timeor end-point measurements of amplification products during PCR (U.S.Pat. Nos. 5,210,015; 5,538,848). The exonuclease assay (Taqman®)employing fluorescent dye-quencher probes (U.S. Pat. No. 5,723,591;Mullah, (1998) “Efficient synthesis of double dye-labelledoligodeoxyribonucleotide probes and their application in a real time PCRassay”, Nucl. Acids Res. 26:1026–1031) gives direct detection ofpolymerase chain reaction (PCR) products in a closed-tube system, withno sample processing beyond that required to perform the PCR. In theTaqman assay, the polymerase that conducts primer extension andamplifies the polynucleotide also displaces and cleaves a probe annealedto target sequence by 5′ to 3′ exonuclease activity. In a Taqman-typeassay, the probe is self-quenching, labelled with fluorescent dye andquencher moieties, either of which may be dyes of the invention.Spectral overlap allows for efficient energy transfer (FRET) when theprobe is intact (Clegg, (1992) “Fluorescence resonance energy transferand nucleic acids”, Meth. Enzymol. 211:353–388). When hybridized to atarget sequence, the probe is cleaved during PCR to release afluorescent signal that is proportional to the amount of target-probehybrid present (U.S. Pat. Nos. 5,538,848; 5,723,591).

The progress of amplification can be monitored continuously, i.e.real-time detection. Spectrally-resolvable atropisomer xanthene dyes ofthe invention are useful in genotyping experiments after PCRamplification of target. In particular, a set of primeroligonucleotides, labelled at the 5′ terminus, each with different dyes,can amplify multiple loci and discriminate single nucleotidepolymorphisms (SNP) and alleles. Electrophoretic separation of thedye-labelled amplification products, with size standards, establishes aprofile or characteristic data set indicating a certain genotypedependent on the set of primer sequences.

V.7A Hybridization Assays

Certain fluorescent dye-quencher probes which hybridize to targetnucleic acids are useful in hybridization assays. When the probe is nothybridized to target, the probe may attain conformations that allowspatial proximity between the fluorescent dye and the quencher moietiesresulting in fluorescence quenching. Upon hybridization to target, themoieties are physically separated, quenching ceases or diminishes, andfluorescence increases. Where the fluorescence is detectable orquantitated, the presence of target sequence in the sample is deduced.The atropisomeric dyes of the invention can also be employed as thefluorescent dye or the quencher moiety. Fluorescent dye-quencher probeswith self-complementary sequences that form a “hairpin” region, socalled “Molecular beacons” (Tyagi and Kramer) undergo the fluorescentchange upon hybridization to their complementary target sequence, e.g.in situ quantitation of mRNA in living cells. Hybridization probeslabelled with different fluorescent dyes, including the atropisomericdyes of the invention, enable multiplex, homogeneous hybridizationassays to be carried out in sealed reaction tubes.

V.8 Chromatography

The aforementioned methods employing substrates labelled withsubstantially pure atropisomer xanthene compounds may also be conductedwhere the labelled substrates are detected by chromatography (HPLC ofMacromolecules, A Practical Approach, Second Edition, R. W. A. Oliver,Ed. (1997) Oxford University Press). The well established techniques ofHPLC enable the separation of large substrates such as polynucleotidesunder reverse phase conditions where the sample substrate is dissolvedand eluted in aqueous organic mobile phase from sorptive ion-exchange orhydrophobic interactions with an immobilized solid phase. When a chiralsubstrate such as a polynucleotide, polypeptide, or polysaccharide islabelled with a racemic mixture of atropisomeric xanthene compounds,diastereomers result. Essentially a redundant set of analytes arecreated which may obscure the analytical result. Analysis of theresulting diastereomeric mixture may lead to double peaks, broad peaks,and other limiting artifacts under the high-resolution conditions ofHPLC. This problem is especially exacerbated where the chiral substrateis a mixture of closely related compounds, such as the nested set ofpolynucleotide fragments generated by the Sanger sequencing method. Useof a substantially pure atropisomeric form of xanthene compounds aslabels for chiral substrates prevents this unwanted hindrance toanalysis by removing one of the diastereomers. The surprising andunexpected benefit of the invention may be exemplified by sharper peaks,less split peaks, and better resolution in general.

V.9 Kits

The invention includes kits comprising the substantially pureatropisomer xanthene compounds of the invention and/or their labelledconjugates. In one embodiment, the kits are useful for conjugating anatropisomer xanthene compound with a linking moiety to another molecule,i.e. a substrate. Such kits generally comprise an atropisomer xantheneof the invention including an optional linking moiety and reagents,enzymes, buffers, solvents, etc. suitable for conjugating the dye toanother molecule or substance. The atropisomer xanthene may be anacceptor or donor of an energy-transfer dye.

In one embodiment, the kits are useful for labelling enzymaticallysynthesized oligonucleotides and polynucleotides with the atropisomerxanthenes of the invention. Such kits generally comprise a labelledenzymatically-incorporatable nucleotide or nucleotide analog accordingto the invention, a mixture of enzymatically-incorporatable nucleotidesor nucleotide analogs capable of supporting continuous primer extensionand a polymerase enzyme. Preferably, the labelledenzymatically-incorporatable nucleotide or nucleotide analog is acompound according to structure IV, most preferably a labelledterminator. Preferred polymerases are thermostable, such as AMPLITAQ®DNA polymerase FS (Applied Biosystems, Foster City, Calif.).

Alternatively, the kit may include one or more primers. The primers maybe labelled with atropisomer xanthenes and energy-transfer dyesincluding atropisomer xanthenes.

V.10 EXAMPLES

The invention will be further clarified by a consideration of thefollowing examples, which are intended to be purely exemplary of thepresent invention and not to in any way limit its scope.

Example 1 Preparation of Menthyl Carbamate Diastereomers of C-1Aminomethyl, C-19 Carboxy Fluorescein, 1a and 1b.

The hydrochloride salt of C-1 aminomethyl, C-19 carboxy fluorescein(5.16 gm, 11.6 mMol, 441.8 MW; Shipchandler (1987) Anal. Biochem.162:89–101) was dissolved in 50 ml of deionized formamide and 10.2 mldiisopropylethylamine. (−) Menthyl chloroformate (3.06 gm, 3.0 ml, 14mMol, 219 MW; Aldrich Chemical, Milwaukee, Wis.; Jour. Chem. Soc., Chem.Commun. (1987) 470; Yodo (1988) Chem. Pharm. Bull. 36:902) was addeddropwise with stirring at room temperature under argon. After 1.5 hours,TLC analysis (ethyl acetate/hexane:4/1) showed partial conversion ofreactant to a higher Rf spot. Another 1 ml (−) Menthyl chloroformate wasadded and stirring was continued for another 0.5 hour. TLC analysisshowed complete conversion to the higher Rf product. Dilution of thereaction mixture with saturated aqueous NaHCO₃ was followed byextraction with 500 ml ethyl acetate. The aqueous fraction was acidifiedto pH 3 and extracted with ethyl acetate. The combined organic fractionswere dried over Na₂SO₄, filtered and concentrated under vacuum to give4.5 gm, 66% yield of a mixture of 1a and 1b as a yellow powder (FIG. 1a).

Example 2 Separation and Isolation of Diastereomers 1a and 1b by HPLC

Crude diastereomers 1a and 1b were separated and purified by a two stagechromatography process on an open column, flash reverse phase columnrough separation, followed by preparative reverse phase HPLC.

The mixture of 1a and 1b was dissolved in ethyl acetate and adsorbed onC-18 reverse phase silica gel. The solvent was removed under vacuum andthe solid was loaded on the top of a pre-equilibrated C-18 reverse phasecolumn (21 cm length×6 cm diameter). The diastereomers were separatedand eluted with 25% CH₃CN in 100 mM TEAA (triethylammonium acetate) bycollecting fractions. The fractions were analyzed by analytical reversephase HPLC on a C-18 column (Metachem ODS3, 25 cm length×4.6 mm innerdiameter) with a linear gradient of 25% to 35% CH₃CN in 100 mM TEAA from0 to 30 minutes at 1.0 ml/min flow rate and 260 nm UV detection. Thefractions that contained the first eluting diastereomer of at least 75%purity were combined and concentrated under vacuum to an orange oil. Thefirst eluting diastereomer was arbitrarily assigned structure 1a. Thefractions that contained the second eluting diastereomer of at least 75%purity were combined and concentrated under vacuum to an orange oil. Thesecond eluting diastereomer was arbitrarily assigned structure 1b.

Diastereomer 1a was purified to 99% isomeric purity by preparativereverse phase HPLC by loading 600–800 mg of 1a purified to 75% purity bythe flash process, dissolved in 500 ml of 100 mM TEAA on to a MetachemODS3 8μ column (Waters Prep LC 2000 System) and eluting under a gradientof 0 to 10% CH₃CN in 100 mM TEAA over 16 min., 10 to 35% CH₃CN over 80min., then hold at 35% CH₃CN for 32 min., at a flow rate of 40 ml/min.,with UV detection at 260 nm. Fractions were collected and analyzed bythe analytical reverse phase HPLC conditions above (FIG. 1 b). Fractionswith isomeric purity of at least 99% were combined, acidified to pH 2with 6N HCl and extracted with ethyl acetate. The ethyl acetate fractionwas washed with saturated NaCl, dried over anhydrous Na₂SO₄,concentrated under vacuum, precipitated with hexane, filtered, and driedto yield 300 to 500 mg of 1a as a bright yellow solid. ¹H NMR(Acetone-d6) δ 9.85, 2H, br; 9.10, 1H, br; 8.35, 1H, d; 8.15, 1H, d;7.83, 1H, s; 7.43, 1H, br; 6.95, 1H, s; 6.70, 4H, m; 4.60, 3H, m; 1.90,2H, m; 1.65, 2H, m; 1.45, 1H, m; 1.30, 2H, m; 0.89, 3H, d; 0.82, 3H, d;0.78, 3H, d. Electrospray Mass Spectroscopy: 610 (M+Na), 588.5 (M+H),

Diastereomer 1b is purified by the same preparative reverse phase HPLCprocess.

Example 3 Synthesis of Atropisomer Amine 2a

Diastereomer 1a (1.1 gm, 1.87 mmoles, 587.6 MW) was dissolved in 100 mlwater and cooled to 0° C. Concentrated sulfuric acid (15 ml) was addeddropwise to give a brownish solution (FIG. 2 a). The temperature wasallowed to rise to room temperature and the mixture was stirredovernight. The mixture was added slowly to 1.5 ml of ice water and thenadsorbed on pre-equilibrated C-18 silica gel (4 cm length×3 cmdiameter). The support was washed with water until the pH of the eluentwas neutral. The crude product was eluted with 200 ml CH₃OH which wasconcentrated under vacuum and dried to yield atropisomer 2a C-1aminomethyl, C-19 carboxy fluorescein sulfate salt as an orange solid(0.93 gm, 95% yield, 503.4 MW). ¹H NMR (methanol-d4) δ 8.43, 1H, d;8.34, 1H, d; 7.92, 1H, s; 7.23, 3H, m; 7.06, 1H, d; 6.98, 1H, d; 4.58,2H, s.

The enantiomeric purity of hydrolyzed and purified 2a was analyzed bychiral column HPLC (Regies (S,S) Whelk-01 10-100 Kromasil FEC column, 25cm length×4.6 mm ID). The sample 2a was dissolved in water and elutedwith a gradient of 0 to 35% ethanol in water containing 0.1% acetic acidover 30 minutes at 1 ml/min. with 254 nm UV detection (FIG. 2 c) anddistinguished from the racemic mixture (FIG. 2 b).

Example 4 Synthesis of Atropisomer Amine 2b

Diastereomer 1b is hydrolyzed, purified, and analyzed to giveatropisomer 2b by the same processes as Example 3 (FIG. 2 a).

Example 5 Synthesis of Atropisomeric Trifluoroacetamide 3a

Atropisomer 2a as the sulfate salt (0.93 gm, 1.84 mmoles, 503.4 MW) wasdissolved in 15 ml ethanol. Triethylamine (1.8 ml, 13 mmoles) and ethyltrifluoroacetate (2.2 ml, 18 mmoles) were slowly added (FIG. 2 a). Themixture was stirred at room temperature under argon for 2.5 hours.Volatiles were removed under vacuum and the resulting residue wasdissolved in 300 ml ethyl acetate and washed with 2×50 ml of 5% HCl. Theethyl acetate fraction was dried over anhydrous Na₂SO₄, filtered andconcentrated under vacuum to yield atropisomeric trifluoroacetamide 3aas an orange solid (0.92 gm, 100% yield, 501.4 MW). ¹H NMR (methanol-d4)δ 8.35, 1H, d; 8.15, 1H, d; 7.80, 1H, s; 6.82, 1H, s; 6.65, 4H, m; 4.82,2H, s.

Example 6 Synthesis of Atropisomeric Trifluoroacetamide 3b

Atropisomer 2b is converted to atropisomeric trifluoroacetamide 3b bythe same process and analyzed by the same methods as Example 5 (FIG. 2a).

Example 7 Synthesis of Atropisomeric NHS Ester 4a

Atropisomeric trifluoroacetamide 3a (0.92 gm, 1.83 mmoles, 501.4),N-hydroxysuccinimide (0.85 gm, 7.3 mmoles), and1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (DAE)(1.05 gm, 5.5 mmoles) were dissolved in 24 ml ethyl acetate and 12 ml of1-methyl-2-pyrrolidinone (NMP) and stirred at room temperature underargon for 2.5 hours (FIG. 3). The mixture was diluted with 300 ml ethylacetate and washed with 2×80 ml of 5% HCl, dried over anhydrous Na₂SO₄,filtered, concentrated under vacuum, and adsorbed on silica gel. Thesilica gel with adsorbed product was loaded on the top of a dry-packedcolumn of silica gel (12 cm length×3 cm ID) and eluted with ethylacetate:hexane/2:1. Fractions containing atropisomeric NHS ester 4a werecollected and combined, concentrated under vacuum and precipitated fromhexane to yield 4a as a bright yellow solid (0.74 gm, 67% yield, 598.4MW). ¹H NMR (methanol-d4) δ 8.42, 1H, d; 8.22, 1H, d; 7.95, 1H, s; 6.81,1H, s; 6.68, 4H, m; 4.82, 2H, s; 2.85, 4H, s.

Example 8 Synthesis of Atropisomeric NHS Ester 4b

Atropisomeric trifluoroacetamide 3b is converted to atropisomeric NHSester 4b by the same process and analyzed by the same methods as Example7 (FIG. 3).

Example 9 Synthesis of2-[(2-Fmoc-aminoethoxy)(hydroxyphosphoryl)oxy]acetic acid NHS 5

Protected phosphodiester linker synthon 5 was prepared by reactingmethyl glycolate 6 (4.5 eq.) with cyclic phosphoramidite Amino-Link™ 7(1 eq.) (Connell (1987) BioTechniques 5:342–348; U.S. Pat. No.4,757,151) and 4-N,N-dimethylaminopyridine (DMAP) (0.1 eq.). The mixturewas stirred at ambient temperature for 1 hour. After the reaction wascomplete (TLC analysis), the solution was cooled with ice-bath and thentreated with a solution of 3-chloroperoxybenzoic acid (4 eq.) inmethylene chloride. The ice-bath was removed. After 30 minutes, anaqueous solution of NaHSO₃ (10%) was added. The mixture was diluted withethyl acetate. The organic layer was washed with NaHSO₃ (10%), saturatedsolution of NaHCO₃, and dried with Na₂SO₄. The crude product waspurified by flash chromatography to afford ester 8, which was heated atreflux for 3 hours (36 mM, 1 eq.) in methylethylketone and NaI (10 eq.).The crude demethylated phosphodiester was dissolved in 0.3 M solution ofLiOH (5 eq.) in H₂O/CH₃OH:1/3) and stirred overnight to cleave themethyl ester. Solvent was removed to afford crude compound 9 which wasthen dissolved in aqueous Na₂CO₃ (5%).N-(9-Fluorenylmethoxy-carbonyloxy)succinimide (FmocOSu, 1.5 eq.) in THFwas added in one portion and stirred at ambient temperature for 3 hours.The crude product was diluted with ethyl acetate and washed with 10%aqueous HCl. The organic layer was dried with Na₂SO₄, filtered,concentrated under vacuum, and purified by flash chromatography toafford Fmoc-acid 10 as a yellow oil.

Fmoc-acid 10 was dissolved in anhydrous CH₂Cl₂ (1 eq.).N-hydroxysuccinimide (4 eq.) was added. The solution was cooled with anice-bath and then treated with dicyclohexyl carbodiimide (DCC, 2 eq.).The ice-bath was then removed, and stirring was continued for 2 hours(with TLC analysis). When the reaction was complete, ethyl acetate wasadded and the solution was washed with 5% aqueous HCl. Removal ofsolvent gave 2-[(2-Fmoc-aminoethoxy)(hydroxyphosphoryl)oxy]acetic acidNHS 5 (FIG. 4).

Example 10 Synthesis of 7-propargylphosphorylamino-7-deaza-ddATP 11

7-Deaza-7-propargylamino-ddATP 12(7-(3-amino-1-propynyl)-2′,3′-dideoxy-7-deazaadenosine-5′-triphosphate;U.S. Pat. Nos. 5,047,519 and 5,151,507) was suspended in 250 mMbicarbonate (pH 9.0) and a solution of2-[(2-Fmoc-aminoethoxy)(hydroxyphosphoryl)oxy]acetic acid NHS 5 in DMSOwas added. After 1 hour, the reaction mixture was purified by HPLC(AX-300 anion exchange). The product fractions were collected,concentrated to dryness, and purified by RP HPLC (C-18 reverse phase) toafford Fmoc-linker ddATP 13. Concentrated ammonium hydroxide (28–30%)was added to Fmoc-linker ddATP 13 and the solution was heated to 55° C.for 20 minutes. Concentration under vacuum gave crude7-propargylphosphorylamino-7-deaza-ddATP 11 which was purified by C-18reverse phase HPLC (FIG. 5).

Example 11 Synthesis of Fmoc-aminomethyl-NHS-FAM 14

Fmoc-aminomethyl-NHS-FAM 14 was prepared by reacting thefluorenylmethoxy-carbonyloxy ester of N-hydroxysuccinimide (Fmoc-OSu)with the HCl salt of p-aminomethylbenzoic acid (both commerciallyavailable) in the presence of base to form the expected N-Fmocderivative. This product was then reacted with N-hydroxysuccinimide inthe presence of DCC to form the NHS ester of the benzoic acid carboxylgroup. This NHS-ester, N-Fmoc derivative of p-aminomethylbenzoic acidhaving the structure:

was then reacted with atropisomer C-1 aminomethyl, C-19 carboxyfluorescein 2a, purified by the method of Example 2, followed byreaction with N-hydroxysuccinimide in the presence of DCC to producesubstantially pure atropisomer, Fmoc-aminomethyl-NHS-FAM 14 (FIG. 6).

Example 12 Synthesis ofAminomethylbenzamide-aminomethyl-FAM-propargylphosphorylamino-ddATP 15

A solution of Fmoc-aminomethyl-NHS-FAM 14 in DMSO was added to7-propargylphosphorylamino-7-deaza-ddATP 11 suspended in 250 mMbicarbonate (pH 9.0). The reaction mixture was placed in the dark atambient temperature for 2 hours. The Fmoc-amino protected product 16 waspurified by HPLC (AX-300 anion exchange), then heated at 55° C. inconcentrated ammonium hydroxide (28–30%) for 20 minutes to hydrolyze theFmoc group. Concentration under vacuum gave crude, substantially pureatropisomer,aminomethylbenzamide-aminomethyl-FAM-propargylphosphorylamino-ddATP 15which was purified by C-18 reverse phase HPLC (FIG. 6).

Example 13 Synthesis of NHS-rhodamine Dye 17

Bicyclic amine 18 (12.8 gm, 47 mmole, U.S. Pat. No. 5,688,808),1-bromo-3-chloropropane (29.3 gm, 187 mmole), sodium iodide (56.4 gm,376 mmole) and sodium bicarbonate (7.9 gm, 94 mmole) was refluxed in 150ml CH₃CN for 18 hours. The mixture was cooled to room temperature,filtered, and evaporated. The filter cake was washed with 300 ml hexanewhich was combined with the filtrate and washed with 2×50 ml water and50 ml saturated NaCl, dried over MgSO₄, filtered, and concentrated undervacuum. The product was purified by chromatography on silica gel,eluting with hexane/ethyl acetate:20/1, to give tricyclic amine pivalateester 19 as a pale yellow oil (9.5 gm, 30 mmole, 64% yield). The esterof 19 was hydrolyzed in a solution of lithium hydroxide monohydrate (2.6gm, 60 mmole) in 15 ml water and 120 ml methanol. After stirring for onehour at room temperature, the mixture was concentrated under vacuum anddissolved in 30 ml 1M HCl which was extracted with 3×100 ml ofdiethylether. The combined ether extracts were washed with 50 ml of 200mM pH 7 phosphate buffer, dried over MgSO₄, filtered and concentratedunder vacuum to give tricyclic amine 20 as a brown solid (FIG. 7).Tricyclic amine 20 and 3,6-dichloro, 4-isopropylcarboxylate phthalicanhydride 21 were refluxed in toluene to give Friedel-Craft acylationproduct ketone 22 (Abs. max 400 nm) (FIG. 8).

Cyclization of 22 with 20 in phosphoryl trichloride and chloroform atreflux gave 23 as a mixture of isopropylcarboxylate regioisomers. Aftercleavage of the isopropyl group, the rhodamine carboxylic acid 24 wasconverted to NHS-rhodamine dye 17 (FIG. 8).

Example 14 Synthesis of Phosphate-Linker, Energy-Transfer TerminatorddATP 25

Aminomethylbenzamide-aminomethyl-FAM-propargylphosphorylamino-ddATP 15from Example 12 was suspended in a solution of 250 mM bicarbonate (pH9.0). A solution of NHS ester 17 (U.S. Pat. No. 5,847,162 for synthesis)in DMSO was added. The reaction mixture was placed in the dark atambient temperature for 2 hours. Purification was done by HPLC, AX-300anion exchange and then C-18 reverse phase to afford pureenergy-transfer ddATP terminator 25 (FIG. 9).

Example 15 Synthesis of Bis-trifluoroacetamide Rhodamine NHS 27

Rhodamine dye 28 was converted to the bis-trifluoroacetamide 29 bytreatment with trifluoroacetic anhydride and triethylamine indiethylether at room temperature. The carboxylic acid was converted tothe NHS ester with dicyclohexylcarbodiimide and N-hydroxysuccinimide togive bis-trifluoroacetamide rhodamine NHS 27 (FIG. 10).

Example 16 Synthesis ofAminomethylbenzamide-aminomethyl-FAM-propargylethoxyamino-ddTTP 32

5-(3-Aminoethoxy-1-propynyl)-2′,3′-dideoxythymidine-5′-triphosphate 30(U.S. Pat. No. 5,821,356) was reacted with substantially pureatropisomer, Fmoc-aminomethyl-NHS-FAM 14 under the same conditions asExample 12 to giveFmoc-aminomethylbenzamide-aminomethyl-FAM-propargylethoxyamino-ddTTP 31which was purified by anion-exchange HPLC. The Fmoc group of 31 wascleaved to give substantially pure atropisomer,aminomethylbenzamide-aminomethyl-FAM-propargylethoxyamino-ddTTP 32 (FIG.11).

Example 17 Synthesis of Energy-Transfer Terminator ddTTP 26

Following the conditions of Example 14, substantially pure atropisomer,aminomethylbenzamide-aminomethyl-FAM-propargylethoxyamino-ddTTP 32 wassuspended in a solution of 250 mM bicarbonate (pH 9.0). A solution ofbis-trifluoroacetamide rhodamine NHS ester 27 in DMSO was added. Thereaction mixture was placed in the dark at ambient temperature for 2hours. Ammonium hydroxide was added to cleave the trifluoroacetamidegroups. Purification was done by HPLC, AX-300 anion exchange and thenC-18 reverse phase to afford pure energy-transfer ddTTP terminator 26(FIG. 12).

Example 18 Sequencing of pGEM with Phosphate-Linker, Energy-TransferTerminator ddATP 25

Following the conditions of U.S. Pat. Nos. 5,770,716; 5,948,648; and6,096,875, the energy-transfer ddATP terminator 25 was used with otherstandard reagents in a Sanger-type, one-color automated DNA sequencingexperiment. The terminator nucleotide 25 was tested as the racemicmixture of atropisomers (top electropherogram) and as a substantiallypure atropisomer (bottom electropherogram) shown in FIGS. 13 a and 13 b.

The dye-terminator sequencing reactions were performed using AmpliTaqDNA Polymerase, FS following protocols provided in the ABI PRISM™ DyeTerminator Cycle Sequencing Core Kit Manual (Applied Biosystems p/n402116). Sequencing of the pGEM-3Zf(+) template was conducted withunlabelled −21 M13 sequencing primer (forward). Reagents, includingbuffer, unlabelled primer, AmpliTaq DNA Polymerase, FS, were from an ABIPRISM™ Dye Terminator Core Kit (Applied Biosystems p/n 402117). The dNTPmix consisted of 2 mM each of dATP, dCTP, dITP, and dUTP or dTTP. Apremix of reaction components was prepared including: 5×Buffer 4.0 μL;dNTP mix 1.0 μL; Template:pGEM®-3Zf(+), 0.2 μg/μL, 2.0 μL; Primer: −21M13 (forward), 0.8 pmol/μL, 4.0 μL; AmpliTaq DNA Polymerase, FS, 0.5 μL;and H₂O 3.5 μL, wherein all quantities are given on a per reactionbasis.

Reactions were assembled in 0.5 ml tubes adapted for the Perkin-Elmer480 DNA Thermal Cycler (Applied Biosystems p/n N801-100 or 0.2 ml tubesfor the Applied Biosystems Gene Amp PCR System 9700). From 1 to 250 pmolof the dye terminator was added to each reaction. 30 μL of mineral oilwas added to the top of each reaction to prevent evaporation (when usingthe Applied Biosystems 480 Thermal Cycler). Reaction volumes were 20 μL,including 15 μL of the above reaction premix, a variable amount of dyelabelled terminator, and a sufficient volume of water to bring the totalreaction volume up to 20 μL. Reactions were thermocycled as follows: 96°C. for 30 sec, 50° C. for 15 sec, and 60° C. for 4 min, for 25 cycles;followed by a 4° C. hold cycle.

All reactions were purified by spin-column purification on Centri-Sepspin columns according to manufacturer's instructions (PrincetonSeparations p/n CS-901). Gel material in the column was hydrated with0.8 mL deionized water for at least 30 minutes at room temperature.After the column was hydrated and it was determined that no bubbles weretrapped in the gel material, the upper and lower end caps of the columnwere removed, and the column was allowed to drain by gravity. The columnwas then inserted into the wash tubes provided in the kit andcentrifuged in a variable speed microcentrifuge at 1300 g for 2 minutes,removed from the wash tube, and inserted into a sample collection tube.The reaction mixture was carefully removed from under the oil and loadedonto the gel material and the tube re-centrifuged. Eluted samples werethen dried in a vacuum centrifuge.

Prior to loading onto a sequencing gel, the dried samples wereresuspended in 25 μL of Template Suppression Reagent (Applied Biosystemsp/n 401674), vortexed, heated to 95° C. for 2 minutes, cooled on ice,vortexed again, and centrifuged (13,000×g). 10 μL of the resuspendedsample was aliquoted into sample vials (Applied Biosystems p/n 401957)adapted for the ABI PRISM™ 310 Genetic Analyzer (Applied Biosystems p/n310-00-100/120). Electrophoresis on the 310 Genetic Analyzer wasperformed with sieving polymers and capillaries specially adapted forDNA sequencing analysis (PE Applied Biosystems p/n 402837 or 4313087(polymer) and p/n 402840 (capillary)). In each case, the sieving polymerincluded nucleic acid denaturants. Samples were electrokineticallyinjected onto the capillary for 30 sec at 2.5 kV, and run for up to 2 hrat 10 to 12.2 kV with the outside wall of the capillary maintained at50° C. to generate electropherograms as sequencing data (FIGS. 13 a–d).

The electropherograms in FIGS. 13 a and 13 b show the specificincorporation of the energy-transfer terminator 25 onto the 3′ terminusof primer extension, polynucleotide fragments during single colorsequencing reactions. The electropherograms plot the fluorescenceintensity emitted (Emission maxima about 650 nm) by the acceptorrhodamine dye of the labelled fragments between about 20 to about 600nucleotides in length as a function of time during an electrophoresisrun on the ABI PRISM™ 310 Genetic Analyzer.

Eluting fragments from 119 to 242 base pairs are plotted in FIG. 13 a.Each of the fragments was 3′ terminated by energy-transfer terminator25. FIG. 13 b shows a more magnified view of fragments 148 to 205 basepairs. The three regions under the arrows in FIGS. 13 a and 13 billustrate the surprising and unexpected improvement in separatingfragments labelled with the substantially pure atropisomer form of 25(bottom panels) relative to the separation of fragments labelled withthe racemic mixture of 25. Better resolution of the fragments wasobserved in the bottom electropherogram with the substantially pureatropisomer than with the racemic mixture of atropisomers in 25. Thelocations marked with arrows are particular loci where substantiallypure atropisomer form of 25 provided the unexpected benefit of betterresolution. By contrast, use of the racemic mixture of atropisomericform of 25 in labelling the chiral primer extension products led todiastereomeric populations of fragments which migrateelectrophoretically at different rates, as exemplified by the broad,overlapping, and split peaks under the arrows in the topelectropherogram.

Additionally, the bottom electropherogram shows more even peak heightsthroughout the sequencing ladder than was observed in the topelectropherogram with the racemic mixture of atropisomers in 25.

Example 19 Sequencing of pGEM with Sulfonate-Linker, Energy-TransferTerminator ddATP 33

Following the general synthesis routes and conditions of the previousExamples, substantially pure atropisomer, sulfonate-linker,energy-transfer terminator ddATP 33 (FIG. 14) was synthesized. Followingthe protocol and conditions of Example 18, 33 was used in single-colorsequencing the pGEM target. Separately, the racemic mixture of 33 wasalso used in the same single-color sequencing experiment. Elutingfragments from 148 to 242 base pairs are plotted in FIG. 13 c. Each ofthe fragments was 3′ terminated by energy-transfer terminator 33. Thethree regions under the arrows in FIG. 13 c illustrate the surprisingand unexpected improvement in separating fragments labelled with thesubstantially pure atropisomer form of 33 (bottom panel) relative to theseparation of fragments labelled with the racemic mixture of 33 (toppanel). Better resolution of the fragments was observed in the bottomelectropherogram with the substantially pure atropisomer than with theracemic mixture of atropisomers in 33.

Example 20 Sequencing of pGEM with Energy-Transfer Terminator ddGTP 34

Following the general synthesis routes and reaction conditions of theprevious Examples, energy-transfer terminator ddGTP 34 (FIG. 15) wassynthesized. The atropisomer forms were separated at the final stage ofsynthesis, i.e. compound 34, by reverse-phase HPLC. Following theprotocol and conditions of Example 18, a substantially pure atropisomerof 34 was used in single-color sequencing the pGEM target. Separately,the racemic mixture of 34 was also used in the same single-colorsequencing experiment. Eluting fragments from 24 to 99 base pairs,detected at about 535 nm, are plotted in FIG. 13 d. Each of thefragments was 3′ terminated by energy-transfer terminator 34. Theregions under the arrows in FIG. 13 d illustrates the surprising andunexpected improvement in separating fragments labelled with thesubstantially pure atropisomer form of 34 (bottom panel) relative to theseparation of fragments labelled with the racemic mixture of 34 (toppanel). Better resolution of virtually every fragment was observed inthe bottom electropherogram with the substantially pure atropisomer thanwith the racemic mixture of atropisomers in 34. The locations markedwith arrows are particular loci where substantially pure atropisomerform of 34 provided the unexpected benefit of better resolution. Everyfragment in the bottom panel, labelled with racemic 34, separates intotwo peaks, detracting from the utility of the data.

All publications cited herein are incorporated by reference, and to thesame extent as if each individual publication was specifically andindividually indicated to be incorporated by reference.

Although only a few embodiments have been described in detail above,those having ordinary skill in the chemical and molecular biology artswill clearly understand that many modifications are possible in theillustrated embodiments without departing from the teachings thereof.All such modifications are intended to be encompassed within thefollowing claims.

1. A method of forming a labelled substrate comprising: reacting asubstrate selected from a polynucleotide, a nucleotide, a nucleoside, apolypeptide, a carbohydrate, a ligand, a substantially enantiomericallypure compound, a particle, and a surface, with the linking moiety of asubstantially pure atropisomer compound having the structure:

wherein Z¹ and Z² are each independently selected from O, OH, NH₂, NHR,and NR₂, X is selected from carboxylate and sulfonate; and at least oneof R¹, R⁴, R⁵, R¹¹, R¹³, R¹⁴, R¹⁷, R¹⁸, R¹⁹, R²⁰, Z¹, or Z² is a linkingmoiety selected from azido, monosubstituted primary amine, disubstitutedsecondary amine, thiol, hydroxyl, halide, epoxide, N-hydroxysuccinimidylester, carboxyl, isothiocyanate, sulfonyl chloride, sulfonate ester,silyl halide, chlorotriazinyl, succinimidyl ester, pentafluorophenylester, maleimide, haloacetyl, epoxide, alkyihalide, allyl halide,aldehyde, ketone, acylazide, anhydride, iodoacetamide, phosphoramiditeand an activated ester, whereby a labelled substrate is formed.
 2. Themethod of claim 1 wherein the linking moiety is N-hydroxysuccinimide. 3.The method of claim 1 wherein the linking moiety is a phosphoramidite.4. The method of claim 1 wherein the substrate is substantiallyenantiomerically pure.
 5. The method of claim 1 wherein thesubstantially enantiomerically pure compound is (+)-menthylchloroformate or (−)-menthyl chloroformate.
 6. The method of claim 1wherein the labelled substrate comprises C-11 aminomethyl, C-19 carboxylfluorescein having the structure:


7. The method of claim 1 wherein the particle is a nanoparticle, amicrosphere, a bead, or a liposome.
 8. The method of claim 1 wherein thesurface is glass.
 9. A method of synthesizing a labelled polynucleotidecomprising: coupling a phosphoramidite compound of the structure:

wherein DYE is a substantially pure atropisomer of a xanthene compoundhaving the structure:

 and aryl-substituted forms thereof, wherein Z¹ and Z² are eachindependently selected from O, OH, NH₂, NHR and NR₂, X is carboxylate orsulfonate; L is a linker; R³⁰ and R³¹ taken separately are selected fromthe group consisting of C₁–C₁₂ alkyl, C₁–C₁₂ cycloalkyl, and aryl; orR³⁰ and R³¹ taken together with the nitrogen atom form a saturatednitrogen heterocycle; and R³² is a phosphite ester protecting group, toa polynucleotide, wherein the polynucleotide is bound to a solidsupport, whereby a labelled polynucleotide is formed.
 10. A method ofseparating atropisomers of a C-11 aminomethyl, C-19 carboxyl fluoresceincompound comprising: (a) reacting a C-11 aminomethyl, C-19 carboxylfluorescein with a substantially pure enantiomer of an active ester orcarboxylic acid to form diastereomeric carbamates; (b) separating thediastereomeric carbamates; and (c) hydrolyzing the separateddiastereomers with aqueous acid.
 11. The method of claim 10 wherein theactive ester is menthyl chioroformate.
 12. The method of claim 10wherein the diastereomeric carbamates are separated by reverse-phaseHPLC.
 13. A method of separating a mixture of labelled substratescomprising: (a) separating a mixture of labelled substrates byelectrophoresis; and (b) detecting the labelled substrates byfluorescence detection, wherein the labelled substrates are comprised ofa substantially pure atropisomer of a xanthene compound having thestructure:

 and aryl-substituted forms thereof, wherein Z¹ and Z² are eachindependently selected from O, OH, NH₂, NHR and NR₂, X is carboxylate orsulfonate.
 14. The method of claim 13 wherein the labelled substratesare labelled polynucleotides.
 15. A method of separating a mixture oflabelled substrates comprising: (a) separating a mixture of labelledsubstrates by chromatography; and (b) detecting the labelled substratesby fluorescence detection, wherein the labelled substrates are comprisedof a substantially pure atropisomer of a xanthene compound having thestructure:

 and aryl-substituted forms thereof, wherein Z¹ and Z² are eachindependently selected from O, OH, NH₂, NHR and NR₂, X is carboxylate orsulfonate.
 16. A method of generating a labelled primer extensionproduct, comprising the step of extending a primer-target hybrid with anenzymatically-incorporatable nucleotide, wherein said primer or saidnucleotide is labelled with a substantially pure atropisomer of axanthene compound having the structure:

and aryl-substituted forms thereof, wherein Z¹ and Z² are eachindependently selected from O, OH, NH₂, NHR and NR2, X is carboxylate orsulfonate, whereby the primer is extended.
 17. The method of claim 16wherein the nucleotide is enzymatically-extendable.
 18. The method ofclaim 16 wherein the primer is a labelled polynucleotide having theformula:

wherein DYE is a substantially pure atropisomer of a xanthene compoundhaving the structure:

 and aryl-substituted forms thereof, wherein Z¹ and Z² are eachindependently selected from O, OH, NH₂, NHR and NR₂, X is carboxylate orsulfonate; B comprises a nucleobase; X is selected from O, NH and S; Lcomprises a linker; R²⁷ is selected from H, OH, halide, azide, amine,alkylamine, C₁–C₆ alkyl, allyl, C₁–C₆ alkoxy, OCH₃, and OCH₂CH═CH₂; andR²⁸ is selected from an intemucleotide phosphodiester and anintemucleotide analog; wherein the polynucleotide comprises 2 to 82nucleotides.
 19. The method of claim 18 wherein B is selected from thegroup consisting of uracil, thymine, cytosine, adenine, 7-deazaadenine,guanine, and 7-deazaguanosine.
 20. The method of claim 16 wherein theenzymatically-incorporatable nucleotide is a labelled nucleoside ornucleotide having the formula:

wherein DYE is a substantially pure atropisomer of a xanthene compoundhaving the structure:

 and aryl-substituted forms thereof, wherein Z¹ and Z² are eachindependently selected from O, OH, NH₂, NHR and NR₂, X is carboxylate orsulfonate; B is a nucleobase; L comprises a linker; R²⁵ is selected fromH, monophosphate, diphosphate, triphosphate, thiophosphate, andphosphate analog; and R²⁶ and R²⁷ are each independently selected from—H, —OH, —F and a moiety which blocks polymerase-mediatedtarget-directed primer extension.
 21. The method of claim 16 furthercomprising a terminator nucleotide.
 22. The method of claim 20 whereinR²⁶ and R²⁷ are —H.
 23. A method of polynucleotide sequencingcomprising: a) forming a mixture of a first, a second, a third, and afourth class of polynucleotides, such that: each polynucleotide in thefirst class includes a 3′-terminal dideoxyadenosine and is labelled witha first dye; each polynucleotide in the second class includes a3′-terminal dideoxycytidine and is labelled with a second dye; eachpolynucleotide in the third class includes a 3′-terminaldideoxyguanosine and is labelled with a third dye; and eachpolynucleotide in the fourth class includes a 3′-terminaldideoxythymidine and is labelled with a fourth dye; wherein at least oneof the first, second, third, or fourth dyes is a substantially pureatropisomer of a xanthene compound having the structure:

and aryl-substituted forms thereof, wherein Z¹ and Z² are eachindependently selected from O, OH, NH₂, NHR and NR₂, X is carboxylate orsulfonate, and the other dyes are spectrally resolvable from each other;and b) separating the polynucleotides on the basis of size.
 24. Themethod of claim 23 further comprising the step of detecting theseparated polynucleotides by fluorescence detection.
 25. The method ofclaim 23 further comprising the step of identifying the 3′-terminalnucleotide of the polynucleotides by the fluorescence spectrum of thedyes.
 26. A method of oligonucleotide ligation, comprising: annealingtwo probes to a target sequence and forming a phosphodiester bondbetween the 5′ terminus of one probe and the 3′ terminus of the otherprobe; wherein one or both probes are labelled with a substantially pureatropisomer of a xanthene compound having the structure:

 and aryl-substituted forms thereof, wherein Z¹ and Z² are eachindependently selected from O, OH, NH₂, NHR and NR₂, X is carboxylate orsulfonate.
 27. A method of fragment analysis comprising: separatinglabelled polynucleotide fragments by a size-dependent separationprocess; and detecting the separated labelled polynucleotide fragmentssubsequent to the separation process, wherein the fragments are labelledwith a substantially pure atropisomer of a xanthene compound having thestructure:

 and aryl-substituted forms thereof, wherein Z¹ and Z² are eachindependently selected from O, OH, NH₂, NHR and NR₂, X is carboxylate orsulfonate.
 28. The method of claim 27 wherein the fragments are labelledwith a mobility-modifying label.
 29. The method of claim 27 wherein thefragments are formed by ligation.
 30. The method of claim 27 wherein thesize-dependent separation process is electrophoresis and the labelledpolynucleotide fragments are detected by fluorescence.
 31. A method ofamplification comprising: annealing two or more primers to a targetpolynucleotide; and extending the primers by a polymerase and a mixtureof enzymatically-extendable nucleotides; wherein at least one of theprimers is a labelled polynucleotide comprising a polynucleotidecovalently attached to a label, wherein the label is a substantiallypure atropisomer of a xanthene compound having the structure:

 and aryl-substituted forms thereof, wherein Z¹ and Z² are eachindependently selected from O, OH, NH₂, NHR and NR₂, X is carboxylate orsulfonate.
 32. A method of amplification comprising: annealing two ormore primers to a target polynucleotide, and extending the primers by apolymerase and a mixture of enzymatically-extendable nucleotides;wherein at least one of the nucleotides is a labelled nucleotide havingthe formula:

wherein DYE is a substantially pure atropisomer of a xanthene compoundhaving the structure:

 and aryl-substituted forms thereof, wherein Z¹ and Z² are eachindependently selected from O, OH, NH₂, NHR and NR₂, X is carboxylate orsulfonate; B comprises a nucleobase; L comprises a linker; R²⁵ isselected from monophosphate, diphosphate, triphosphate, thiophosphate,and phosphate analog; and R²⁶ and R²⁷, when taken alone, are eachindependently selected from —H, —OH, —F and a moiety that blockspolymerase-mediated target-directed primer extension.
 33. A method ofamplification comprising: annealing two or more primers and afluorescent dye-quencher probe to a target nucleic acid; and extendingthe primers by polymerase and a mixture of enzymatically-extendablenucleotides; wherein the probe is a labelled polynucleotide comprising apolynucleotide covalently attached to a label, wherein the label is asubstantially pure atropisomer of a xanthene compound having thestructure:

 and aryl-substituted forms thereof, wherein Z¹ and Z² are eachindependently selected from O, OH, NH₂, NHR and NR₂, X is carboxylate orsulfonate.